Fuel compositions comprising isoprene derivatives

ABSTRACT

The invention provides for methods, compositions and systems using bioisoprene derived from renewable carbon for production of a variety of hydrocarbon fuels and fuel additives.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 61/187,959, filed Jun. 17, 2009, the disclosure of which isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

The development of renewable transportation fuels is one of the keychallenges of the twenty-first century. The current market is dominatedby ethanol derived from yeast fermentation of sucrose and starch, and toa lesser extent by biodiesel (fatty acid esters) derived fromtriglycerides. Ethanol has limitations as a liquid fuel with a lowerenergy density relative to hydrocarbons. In addition, ethanol cannot betransported in conventional infrastructure due to its affinity for waterand corrosive nature. Processes for the conversion of renewable carbonsources (biomass, sugars, oils) to hydrocarbon fuels offer an attractivealternative to bioethanol.

Isoprene (2-methyl-1,3-butadiene) is a key industrial chemical usedprimarily for the production of synthetic rubber. Currently isoprene isderived from petrochemical sources either directly by cracking ofnaphtha and other light petroleum fractions, or indirectly throughchemical synthesis (See, for examples, H. Pommer and A. Nurrenbach,Industrial Synthesis of Terpene Compounds, Pure Appl. Chem., 1975, 43,527-551; H. M. Weitz and E. Loser, Isoprene, in Ullmann's Encyclopediaof Industrial Chemistry, Seventh Edition, Electronic Release, Wiley-VCHVerlag GMBH, Weinheim, 2005; and H. M. Lybarger, Isoprene in Kirk-OthmerEncyclopedia of Chemical Technology, 4th ed., Wiley, New York (1995),14, 934-952.) The resulting crude isoprene streams are typicallysubjected to extensive purification processes in order to removenumerous chemically similar impurities, many of which can interfere withsubsequent transformation of isoprene to polymers and other chemicals.

In contrast, isoprene derived from biological sources contains very fewhydrocarbon impurities and instead contains a number of oxygenatedcompounds such as ethanol, acetaldehyde and acetone. Many of thesecompounds can be easily removed by contact with water or passage throughalumina or other adsorbents.

Industry relies on petrochemical feedstocks for isoprene production andextensive purification trains are needed before isoprene can beconverted to polymers and other chemicals. Cost effective methods aredesirable for converting biologically produced isoprene to valuablechemical products taking advantage of the high purity and/or the uniqueimpurity profiles of bioisoprene.

All patents, patent applications, documents, and articles cited hereinare herein incorporated by reference in their entirety.

BRIEF SUMMARY OF THE INVENTION

Disclosed are methods and systems for producing fuel constituents fromhighly pure isoprene and fuel compositions produced from highly pureisoprene.

In one aspect, the invention provides a method for producing a fuelconstituent from a bioisoprene composition comprising chemicallytransforming a substantial portion of the isoprene in the bioisoprenecomposition to non-isoprene compounds. In one embodiment, thebioisoprene composition is chemically transformed by subjecting thebioisoprene composition to heat or catalytic conditions suitable forisoprene dimerization to produce an isoprene dimer and thencatalytically hydrogenating the isoprene dimer to form a saturated C10fuel constituent. In another embodiment, the bioisoprene composition ischemically transformed by (i) partially hydrogenating the bioisoprenecomposition to produce an isoamylene, (ii) dimerizing the isoamylenewith a mono-olefin selected from the group consisting of isoamylene,propylene and isobutene to form a dimate and (iii) completelyhydrogenating the dimate to produce a fuel constituent. In someembodiments, at least about 95% of isoprene in the bioisoprenecomposition is converted to non-isoprene compounds during the chemicaltransformation. In some embodiments, the bioisoprene composition isheated to about 150° C. to about 250° C. to produce an unsaturatedcyclic isoprene dimer and the unsaturated cyclic isoprene dimer ishydrogenated catalytically to produce a saturated cyclic isoprene dimerfuel constituent. In some embodiments, the method comprises: (i)contacting the bioisoprene composition with a catalyst for catalyzingcyclo-dimerization of isoprene to produce an unsaturated cyclic isoprenedimer and the unsaturated cyclic isoprene dimer is hydrogenatedcatalytically to produce a saturated cyclic isoprene dimer fuelconstituent. In some embodiments, the catalyst for catalyzingcyclo-dimerization of isoprene comprising a catalyst selected from thegroup consisting of a nickel catalyst, iron catalysts and chromiumcatalysts. In some embodiments, the step of partially hydrogenating thebioisoprene composition comprises contacting the bioisoprene compositionwith hydrogen gas and a catalyst for catalyzing partial hydrogenation ofisoprene. In some embodiments, the catalyst for catalyzing partialhydrogenation of isoprene comprises a palladium catalyst. In someembodiments, the step of dimerizing the isoamylene with a mono-olefincomprises contacting the isoamylene with the mono-olefin in the presenceof a catalyst for catalyzing dimerization of mono-olefin. In someembodiments, the catalyst for catalyzing dimerization of mono-olefincomprises an acid catalyst. In some embodiments, the method furthercomprises purifying the isoprene from the bioisoprene composition priorto chemically transforming the bioisoprene composition to a fuelconstituent.

In one aspect, the invention provides a system for producing a fuelconstituent from a bioisoprene composition, wherein a substantialportion of the isoprene in the bioisoprene composition is chemicallyconverted to non-isoprene compounds, the system comprising a bioisoprenecomposition and (a) (i) one or more chemicals capable of dimerizingisoprene in the bioisoprene composition or a source of heat capable ofdimerizing isoprene in the bioisoprene composition; and (ii) a catalystcapable of hydrogenating the isoprene dimer to form a saturated C10 fuelconstituent; or (b) (i) a chemical capable of partially hydrogenatingisoprene in the bioisoprene composition to produce an isoamylene, (ii) achemical capable of dimerizing the isoamylene with mono-olefins selectedfrom the group consisting of isoamylene, propylene and isobutene to forma dimate and (iii) a chemical capable of completely hydrogenating thedimate to produce a fuel constituent.

In some embodiments of the system, the bioisoprene compositioncomprising greater than about 2 mg of isoprene and comprising greaterthan or about 99.94% isoprene by weight compared to the total weight ofall C5 hydrocarbons in the composition. In some embodiments of thesystem, the one or more chemicals capable of dimerizing isoprenecomprises catalyst for catalyzing cyclo-dimerization of isoprenecomprising a catalyst selected from the group consisting of rutheniumcatalysts, nickel catalysts, iron catalysts and chromium catalysts. Insome embodiments of the system, the catalyst for hydrogenating theunsaturated isoprene dimers comprises a catalyst selected from the groupconsisting of palladium catalysts, nickel catalysts, ruthenium catalystsand rhodium catalysts. In some embodiments of the system, the chemicalcapable of partially hydrogenating isoprene comprises a palladiumcatalyst. In some embodiments of the system, the chemical capable ofdimerizing the isoamylene with mono-olefins comprises an acid catalyst.

In one aspect, the invention provides a fuel composition comprising afuel constituent produced by the methods described herein. In someembodiments, the fuel composition is substantially free of isoprene. Insome embodiments, the fuel composition has δ¹³C value which is greaterthan −22‰ or within the range of −32‰ to −24%0.

In some aspects, the invention provides a system for producing a fuelconstituent from isoprene comprising: (a) a commercially beneficialamount of highly pure isoprene; and (b) a fuel constituent produced fromat least a portion of the highly pure isoprene; wherein at least aportion of the commercially beneficial amount of highly pure isopreneundergoes a chemical transformation.

In some embodiments of the system, the commercially beneficial amount ofhighly pure isoprene comprises greater than about 2 mg of isoprene andcomprising greater than or about 99.94% isoprene by weight compared tothe total weight of all C5 hydrocarbons in the composition. In someembodiments, the commercially beneficial amount of highly pure isoprenecomprises greater than about 2 mg of isoprene and comprising one or morecompounds selected from the group consisting of ethanol, acetone, C5prenyl alcohols, and isoprenoid compounds with 10 or more carbon atoms.In some embodiments, the commercially beneficial amount of highly pureisoprene comprises greater than about 2 mg of isoprene and comprisingone or more second compounds selected from the group consisting ofethanol, acetone, methanol, acetaldehyde, methacrolein, methyl vinylketone, 2-methyl-2-vinyloxirane, cis- and trans-3-methyl-1,3-pentadiene,a C5 prenyl alcohol, 2-heptanone, 6-methyl-5-hepten-2-one,2,4,5-trimethylpyridine, 2,3,5-trimethylpyrazine, citronellal,methanethiol, methyl acetate, 1-propanol, diacetyl, 2-butanone,2-methyl-3-buten-2-ol, ethyl acetate, 2-methyl-1-propanol,3-methyl-1-butanal, 3-methyl-2-butanone, 1-butanol, 2-pentanone,3-methyl-1-butanol, ethyl isobutyrate, 3-methyl-2-butenal, butylacetate, 3-methylbutyl acetate, 3-methyl-3-buten-1-yl acetate,3-methyl-2-buten-1-yl acetate, 3-hexen-1-ol, 3-hexen-1-yl acetate,limonene, geraniol (trans-3,7-dimethyl-2,6-octadien-1-ol), citronellol(3,7-dimethyl-6-octen-1-ol), (E)-3,7-dimethyl-1,3,6-octatriene,(Z)-3,7-dimethyl-1,3,6-octatriene, and 2,3-cycloheptenolpyridine;wherein the amount of the second compound relative to the amount of theisoprene is greater than or about 0.01% (w/w). In some embodiments, thecommercially beneficial amount of highly pure isoprene comprises greaterthan about 2 mg of isoprene and comprising less than or about 0.5 μg/Lper compound for any compound in the composition that inhibits thepolymerization of isoprene. In some preferred embodiments, thecommercially beneficial amount of highly pure isoprene is produced by abiological process.

In some embodiments of the system, the fuel constituent comprises one ormore compounds selected from the group consisting of cyclic isoprenedimers and trimers, linear isoprene oligomers, aromatic and alicyclicisoprene derivatives, and oxygenated isoprene derivatives. In someembodiments, the oxygenated isoprene derivatives are compounds selectedfrom the group consisting of alcohols, ketones, esters and ethersderived from isoprene.

In some embodiments, the fuel constituent comprises cyclic isoprenedimers and the chemical transformation comprises a dimerization reactionof isoprene. In some embodiments, the dimerization reaction is carriedout by heating the commercially beneficial amount of highly pureisoprene. In some embodiments, the dimerization reaction of isopreneproduces a product comprising unsaturated isoprene dimers and thechemical transformation further comprises a hydrogenation reaction ofthe unsaturated isoprene dimers. In some embodiments, the system furthercomprises a catalyst for catalyzing the hydrogenation reaction of theunsaturated isoprene dimers. In some embodiments, the catalyst forcatalyzing the hydrogenation reaction of the unsaturated isoprene dimerscomprising a catalyst selected from the group consisting of palladiumcatalysts, nickel catalysts, ruthenium catalysts and rhodium catalysts.

In some embodiments, the dimerization reaction is carried out bycontacting the commercially beneficial amount of highly pure isoprenewith a catalyst for catalyzing cyclo-dimerization of isoprene. In someembodiments, the catalyst for catalyzing cyclo-dimerization of isoprenecomprising a catalyst selected from the group consisting of rutheniumcatalysts, nickel catalysts, iron catalysts and chromium catalysts. Insome embodiments, the catalyst for catalyzing cyclo-dimerization ofisoprene is a nickel catalyst and the fuel constituent comprising one ormore eight-membered ring dimers of isoprene.

In some embodiments, the fuel constituent comprises linear and/or cyclictrimers of isoprene and the chemical transformation comprises catalytictrimerization of isoprene.

In one aspect, the invention provides a method for producing a fuelconstituent from isoprene comprising: (a) obtaining a commerciallybeneficial amount of highly pure isoprene; and (b) chemicallytransforming at least a portion of the commercially beneficial amount ofhighly pure isoprene to a fuel constituent. In some embodiments, thecommercially beneficial amount of highly pure isoprene comprisesbioisoprene.

In some embodiments, the commercially beneficial amount of highly pureisoprene is obtained by the steps comprising: (i) culturing cellscomprising a heterologous nucleic acid encoding an isoprene synthasepolypeptide under suitable culture conditions for the production ofisoprene, wherein the cells (1) produce greater than about 400nmole/g_(wcm)/hr of isoprene, (2) convert more than about 0.002 molarpercent of the carbon that the cells consume from a cell culture mediuminto isoprene, or (3) have an average volumetric productivity ofisoprene greater than about 0.1 mg/L_(broth)/hr of isoprene, and (ii)producing isoprene. In some embodiments, the cells further comprise aheterologous nucleic acid encoding an isoprene synthase polypeptide oran MVA pathway polypeptide.

In some embodiments of the methods described herein, chemicallytransforming at least a portion of the commercially beneficial amount ofhighly pure isoprene to a fuel constituent comprises: (i) heating thecommercially beneficial amount of highly pure isoprene to about 150° C.to about 250° C.; (ii) converting at least a portion of the commerciallybeneficial amount of highly pure isoprene to unsaturated cyclic isoprenedimers; (iii) hydrogenating the unsaturated cyclic isoprene dimers toproduce saturated cyclic isoprene dimers; and (iv) producing the fuelconstituent. In some embodiments, at least about 20% to about 100% ofisoprene in the commercially beneficial amount of highly pure isopreneis converted to unsaturated cyclic isoprene dimers.

In some embodiments of the method, chemically transforming at least aportion of the commercially beneficial amount of highly pure isoprene toa fuel constituent comprises: (i) contacting the commercially beneficialamount of highly pure isoprene with a catalyst for catalyzingcyclo-dimerization of isoprene, (ii) converting at least a portion ofthe commercially beneficial amount of highly pure isoprene to cyclicisoprene dimers; and (iii) producing the fuel constituent.

In some embodiments of the method, chemically transforming at least aportion of the commercially beneficial amount of highly pure isoprene toa fuel constituent comprises: (i) contacting the commercially beneficialamount of highly pure isoprene with a catalyst for catalyzingcyclo-dimerization of isoprene, (ii) converting at least a portion ofthe commercially beneficial amount of highly pure isoprene tounsaturated cyclic isoprene dimers; (iii) hydrogenating the unsaturatedcyclic isoprene dimers to produce saturated cyclic isoprene dimers; and(iv) producing the fuel constituent. In some embodiments of the method,the catalyst for catalyzing cyclo-dimerization of isoprene comprising acatalyst selected from the group consisting of a nickel catalyst, ironcatalysts and chromium catalysts.

In some embodiments of the method, chemically transforming at least aportion of the commercially beneficial amount of highly pure isoprene toa fuel constituent comprises: (i) contacting the highly pure isoprenecomposition with a catalyst system; (ii) converting at least a portionof the starting isoprene composition to unsaturated isoprene dimersand/or trimers; and (iii) hydrogenating the unsaturated dimers and/ortrimers to produce saturated C10 and/or C15 hydrocarbons.

In some embodiments, the method for producing a fuel constituent fromisoprene comprises: (a) obtaining a commercially beneficial amount ofany of the highly pure isoprene starting composition described herein;(b) converting at least a portion of the starting isoprene compositionto oxygenated isoprene derivatives; and optionally (c) hydrogenating anyunsaturated oxygenated isoprene derivatives to produce saturatedoxygenates. In some embodiments, the oxygenated isoprene derivatives arecompounds selected from the group consisting of alcohols, ketones,esters and ethers derived from isoprene.

In some embodiments, any of the methods described herein furthercomprises purifying the commercially beneficial amount of highly pureisoprene prior to chemically transforming at least a portion of thecommercially beneficial amount of highly pure isoprene to a fuelconstituent.

In one aspect, provided is a continuous process for producing a fuelconstituent from isoprene comprising: (a) continuously producing acommercially beneficial amount of highly pure isoprene; and (b)continuously transforming chemically at least a portion of thecommercially beneficial amount of highly pure isoprene to a fuelconstituent. In some embodiments, the commercially beneficial amount ofhighly pure isoprene comprising a gas phase comprising isoprene. In someembodiments, the method further comprises passing the gas phasecomprising isoprene to a reactor for chemically transforming at least aportion of the commercially beneficial amount of highly pure isoprene toa fuel constituent. In a preferred embodiment, the commerciallybeneficial amount of highly pure isoprene comprises bioisoprene.

Also provided is a fuel composition comprising a fuel constituentproduced by any of the methods described herein. In some embodiments,the fuel constituent comprises less than or about 0.5 μg/L a productfrom a C5 hydrocarbon other than isoprene after undergoing the stepsaccording to the methods described herein. In some embodiments, the fuelconstituent comprises one or more product from one or more compoundselected from the group consisting of ethanol, acetone, methanol,acetaldehyde, methacrolein, methyl vinyl ketone,2-methyl-2-vinyloxirane, cis- and trans-3-methyl-1,3-pentadiene, a C5prenyl alcohol, 2-heptanone, 6-methyl-5-hepten-2-one,2,4,5-trimethylpyridine, 2,3,5-trimethylpyrazine, citronellal,methanethiol, methyl acetate, 1-propanol, diacetyl, 2-butanone,2-methyl-3-buten-2-ol, ethyl acetate, 2-methyl-1-propanol,3-methyl-1-butanal, 3-methyl-2-butanone, 1-butanol, 2-pentanone,3-methyl-1-butanol, ethyl isobutyrate, 3-methyl-2-butenal, butylacetate, 3-methylbutyl acetate, 3-methyl-3-buten-1-yl acetate,3-methyl-2-buten-1-yl acetate, 3-hexen-1-ol, 3-hexen-1-yl acetate,limonene, geraniol(trans-3,7-dimethyl-2,6-octadien-1-ol), citronellol(3,7-dimethyl-6-octen-1-ol), (E)-3,7-dimethyl-1,3,6-octatriene,(Z)-3,7-dimethyl-1,3,6-octatriene and 2,3-cycloheptenolpyridine afterundergoing the steps according to the methods described herein.

In some embodiments, the fuel composition comprises a fuel compositionhaving δ¹³C value which is greater than −22‰. In some embodiments, thefuel composition has δ¹³C value which is within the range of −22‰ to−10‰ or −34‰ to −24‰. In some embodiments, the fuel composition hasf_(m) value which is greater than 0.9. Also provided is a blend of anyof the fuel compositions described herein with a petroleum based fuel inthe amount of from about 1% to about 95% by weight or volume, based onthe total weight or volume of the total fuel composition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the nucleotide sequence of a kudzu isoprene synthase genecodon-optimized for expression in E. coli (SEQ ID NO:1). The atg startcodon is in italics, the stop codon is in bold and the added PstI siteis underlined.

FIG. 2 is a map of pTrcKudzu.

FIGS. 3A-C are the nucleotide sequence of pTrcKudzu (SEQ ID NO:2). TheRBS is underlined, the kudzu isoprene synthase start codon is in boldcapitol letters and the stop codon is in bold, capital letters. Thevector backbone is pTrcHis2B.

FIG. 4 is a map of pETNHisKudzu.

FIGS. 5A-C are the nucleotide sequence of pETNHisKudzu (SEQ ID NO:3).

FIG. 6 is a map of pCL-lac-Kudzu.

FIGS. 7A-C are the nucleotide sequence of pCL-lac-Kudzu (SEQ ID NO:4).

FIG. 8A is a graph showing the production of isoprene in E. coli BL21cells with no vector.

FIG. 8B is a graph showing the production of isoprene in E. coli BL21cells with pCL-lac-Kudzu

FIG. 8C is a graph showing the production of isoprene in E. coli BL21cells with pTrcKudzu.

FIG. 8D is a graph showing the production of isoprene in E. coli BL21cells with pETN-HisKudzu.

FIG. 9A is a graph showing OD over time of fermentation of E. coliBL21/pTrcKudzu in a 14 liter fed batch fermentation.

FIG. 9B is a graph showing isoprene production over time of fermentationof E. coli BL21/pTrcKudzu in a 14 liter fed batch fermentation.

FIG. 10A is a graph showing the production of isoprene in Panteoacitrea. Control cells without recombinant kudzu isoprene synthase. Greydiamonds represent isoprene synthesis, black squares represent OD₆₀₀.

FIG. 10B is a graph showing the production of isoprene in Panteoa citreaexpressing pCL-lac Kudzu. Grey diamonds represent isoprene synthesis,black squares represent OD₆₀₀.

FIG. 10C is a graph showing the production of isoprene in Panteoa citreaexpressing pTrcKudzu. Grey diamonds represent isoprene synthesis, blacksquares represent OD₆₀₀.

FIG. 11 is a graph showing the production of isoprene in Bacillussubtilis expressing recombinant isoprene synthase. BG3594comK is a B.subtilis strain without plasmid (native isoprene production). CF₄₄₃ isB. subtilis strain BG3594comK with pBSKudzu (recombinant isopreneproduction). IS on the y-axis indicates isoprene.

FIGS. 12A-C are the nucleotide sequence of pBS Kudzu #2 (SEQ ID NO:5).

FIG. 13 is the nucleotide sequence of kudzu isoprene synthasecodon-optimized for expression in Yarrowia (SEQ ID NO:6).

FIG. 14 is a map of pTrex3g comprising a kudzu isoprene synthase genecodon-optimized for expression in Yarrowia.

FIGS. 15A-C are the nucleotide sequence of vector pSPZ1(MAP29Spb) (SEQID NO:7).

FIG. 16 is the nucleotide sequence of the synthetic kudzu (Puerariamontana) isoprene gene codon-optimized for expression in Yarrowia (SEQID NO:8).

FIG. 17 is the nucleotide sequence of the synthetic hybrid poplar(Populus alba×Populus tremula) isoprene synthase gene (SEQ ID NO:9). TheATG start codon is in bold and the stop codon is underlined.

FIG. 18A (FIGS. 18A1 and 18A2) shows a schematic outlining constructionof vectors pYLA 1, pYL1 and pYL2 (SEQ ID NO:75, 73, 72, 71, 70, 69).

FIG. 18B shows a schematic outlining construction of the vectorpYLA(POP1) (SEQ ID NO:68, 69).

FIG. 18C shows a schematic outlining construction of the vectorpYLA(KZ1)

FIG. 18D shows a schematic outlining construction of the vectorpYLI(KZ1) (SEQ ID NO:66, 67)

FIG. 18E shows a schematic outlining construction of the vectorpYLI(MAP29)

FIG. 18F shows a schematic outlining construction of the vectorpYLA(MAP29)

FIG. 19A shows the MVA and DXP metabolic pathways for isoprene (based onF. Bouvier et al., Progress in Lipid Res. 44: 357-429, 2005). Thefollowing description includes alternative names for each polypeptide inthe pathways and a reference that discloses an assay for measuring theactivity of the indicated polypeptide (each of these references are eachhereby incorporated by reference in their entireties, particularly withrespect to assays for polypeptide activity for polypeptides in the MVAand DXP pathways). Mevalonate Pathway: AACT; Acetyl-CoAacetyltransferase, MvaE, EC 2.3.1.9. Assay: J. Bacteriol., 184:2116-2122, 2002; HMGS; Hydroxymethylglutaryl-CoA synthase, MvaS, EC2.3.3.10. Assay: J. Bacteriol., 184: 4065-4070, 2002; HMGR;3-Hydroxy-3-methylglutaryl-CoA reductase, MvaE, EC 1.1.1.34. Assay: J.Bacteriol., 184: 2116-2122, 2002; MVK; Mevalonate kinase, ERG12, EC2.7.1.36. Assay: Curr Genet. 19:9-14, 1991. PMK; Phosphomevalonatekinase, ERGS, EC 2.7.4.2, Assay: Mol Cell Biol., 11:620-631, 1991;DPMDC; Diphosphomevalonate decarboxylase, MVD1, EC 4.1.1.33. Assay:Biochemistry, 33:13355-13362, 1994; IDI; Isopentenyl-diphosphatedelta-isomerase, IDI1, EC 5.3.3.2. Assay: J. Biol. Chem.264:19169-19175, 1989. DXP Pathway: DXS; 1-Deoxyxylulose-5-phosphatesynthase, dxs, EC 2.2.1.7. Assay: PNAS, 94:12857-62, 1997; DXR;1-Deoxy-D-xylulose 5-phosphate reductoisomerase, dxr, EC 2.2.1.7. Assay:Eur. J. Biochem. 269:4446-4457, 2002; MCT;4-Diphosphocytidyl-2C-methyl-D-erythritol synthase, IspD, EC 2.7.7.60.Assay: PNAS, 97: 6451-6456, 2000; CMK;4-Diphosphocytidyl-2-C-methyl-D-erythritol kinase, IspE, EC 2.7.1.148.Assay: PNAS, 97:1062-1067, 2000; MCS; 2C-Methyl-D-erythritol2,4-cyclodiphosphate synthase, IspF, EC 4.6.1.12. Assay: PNAS,96:11758-11763, 1999; HDS; 1-Hydroxy-2-methyl-2-(E)-butenyl4-diphosphate synthase, ispG, EC 1.17.4.3. Assay: J. Org. Chem.,70:9168-9174, 2005; HDR; 1-Hydroxy-2-methyl-2-(E)-butenyl 4-diphosphatereductase, IspH, EC 1.17.1.2. Assay: JACS, 126:12847-12855, 2004.

FIG. 19B illustrates the classical and modified MVA pathways. 1,acetyl-CoA acetyltransferase (AACT); 2, HMG-CoA synthase (HMGS); 3,HMG-CoA reductase (HMGR); 4, mevalonate kinase (MVK); 5,phosphomevalonate kinase (PMK); 6, diphosphomevalonate decarboxylase(MVD or DPMDC); 7, isopentenyl diphosphate isomerase (IDI); 8,phosphomevalonate decarboxylase (PMDC); 9, isopentenyl phosphate kinase(IPK). The classical MVA pathway proceeds from reaction 1 throughreaction 7 via reactions 5 and 6, while a modified MVA pathway goesthrough reactions 8 and 9. P and PP in the structural formula arephosphate and pyrophosphate, respectively. This figure was taken fromKoga and Morii, Microbiology and Mol. Biology. Reviews, 71:97-120, 2007,which is incorporated by reference in its entirety, particular withrespect to nucleic acids and polypeptides of the modified MVA pathway.The modified MVA pathway is present, for example, in some archaealorganisms, such as Methanosarcina mazei.

FIG. 20 (FIGS. 20A and 20B) shows graphs representing results of theGC-MS analysis of isoprene production by recombinant Y. lipolyticastrains without (left) or with (right) a kudzu isoprene synthase gene.The arrows indicate the elution time of the authentic isoprene standard.

FIG. 21 is a map of pTrcKudzu yIDI DXS Kan.

FIGS. 22A-D are the nucleotide sequence of pTrcKudzu yIDI DXS Kan (SEQID NO:10).

FIG. 23A is a graph showing production of isoprene from glucose inBL21/pTrcKudzukan. Time 0 is the time of induction with IPTG (400 μmol).The x-axis is time after induction; the y-axis is OD₆₀₀ and the y2-axisis total productivity of isoprene (μg/L headspace or specificproductivity (μg/L headspace/OD). Diamonds represent OD₆₀₀, circlesrepresent total isoprene productivity (μg/L) and squares representspecific productivity of isoprene (μg/L/OD).

FIG. 23B is a graph showing production of isoprene from glucose inBL21/pTrcKudzu yIDI kan. Time 0 is the time of induction with IPTG (400μmol). The x-axis is time after induction; the y-axis is OD₆₀₀ and they2-axis is total productivity of isoprene (μg/L headspace or specificproductivity (μg/L headspace/OD). Diamonds represent OD₆₀₀, circlesrepresent total isoprene productivity (μg/L) and squares representspecific productivity of isoprene (μg/L/OD).

FIG. 23C is a graph showing production of isoprene from glucose inBL21/pTrcKudzu DXS kan. Time 0 is the time of induction with IPTG (400μmol). The x-axis is time after induction; the y-axis is OD₆₀₀ and they2-axis is total productivity of isoprene (μg/L headspace or specificproductivity (μg/L headspace/OD). Diamonds represent OD₆₀₀, circlesrepresent total isoprene productivity (μg/L) and squares representspecific productivity of isoprene (μg/L/OD).

FIG. 23D is a graph showing production of isoprene from glucose inBL21/pTrcKudzu yIDI DXS kan. Time 0 is the time of induction with IPTG(400 μmol). The x-axis is time after induction; the y-axis is OD₆₀₀ andthe y2-axis is total productivity of isoprene (μg/L headspace orspecific productivity (μg/L headspace/OD). Diamonds represent OD₆₀₀,circles represent total isoprene productivity (μg/L) and squaresrepresent specific productivity of isoprene (μg/L/OD).

FIG. 23E is a graph showing production of isoprene from glucose inBL21/pCL PtrcKudzu. Time 0 is the time of induction with IPTG (400μmol). The x-axis is time after induction; the y-axis is OD₆₀₀ and they2-axis is total productivity of isoprene (μg/L headspace or specificproductivity (μg/L headspace/OD). Diamonds represent OD₆₀₀, circlesrepresent total isoprene productivity (μg/L) and squares representspecific productivity of isoprene (μg/L/OD).

FIG. 23F is a graph showing production of isoprene from glucose inBL21/pCL PtrcKudzu yIDI. Time 0 is the time of induction with IPTG (400μmol). The x-axis is time after induction; the y-axis is OD₆₀₀ and they2-axis is total productivity of isoprene (μg/L headspace or specificproductivity (μg/L headspace/OD). Diamonds represent OD₆₀₀, circlesrepresent total isoprene productivity (μg/L) and squares representspecific productivity of isoprene (μg/L/OD).

FIG. 23G is a graph showing production of isoprene from glucose inBL21/pCL PtrcKudzu DXS. Time 0 is the time of induction with IPTG (400μmol). The x-axis is time after induction; the y-axis is OD₆₀₀ and they2-axis is total productivity of isoprene (μg/L headspace or specificproductivity (μg/L headspace/OD). Diamonds represent OD₆₀₀, circlesrepresent total isoprene productivity (μg/L) and squares representspecific productivity of isoprene (μg/L/OD).

FIG. 23H is a graph showing production of isoprene from glucose inBL21/pTrcKudzuIDIDXSkan. The arrow indicates the time of induction withIPTG (400 μmol). The x-axis is time after induction; the y-axis is OD₆₀₀and the y2-axis is total productivity of isoprene (μg/L headspace orspecific productivity (μg/L headspace/OD). Black diamonds representOD₆₀₀, black triangles represent isoprene productivity (μg/L) and whitesquares represent specific productivity of isoprene (μg/L/OD).

FIG. 24 is a map of pTrcKKDyIkIS kan.

FIGS. 25A-D are a nucleotide sequence of pTrcKKDyIkIS kan (SEQ IDNO:11).

FIG. 26 is a map of pCL PtrcUpperPathway.

FIGS. 27A-27D is a nucleotide sequence of pCL PtrcUpperPathway (SEQ IDNO:12).

FIG. 28 shows a map of the cassette containing the lower MVA pathway andyeast idi for integration into the B. subtilis chromosome at the nprElocus. nprE upstream/downstream indicates 1 kb each of sequence from thenprE locus for integration. aprE promoter (alkaline serine proteasepromoter) indicates the promoter (−35, −10, +1 transcription start site,RBS) of the aprE gene. MVK1 indicates the yeast mevalonate kinase gene.RBS-PMK indicates the yeast phosphomevalonate kinase gene with aBacillus RBS upstream of the start site. RBS-MPD indicates the yeastdiphosphomevalonate decarboxylase gene with a Bacillus RBS upstream ofthe start site. RBS-IDI indicates the yeast idi gene with a Bacillus RBSupstream of the start site. Terminator indicates the terminator alkalineserine protease transcription terminator from B. amyliquefaciens. SpecRindicates the spectinomycin resistance marker. “nprE upstream repeat foramp.” indicates a direct repeat of the upstream region used foramplification.

FIGS. 29A-D are a nucleotide sequence of cassette containing the lowerMVA pathway and yeast idi for integration into the B. subtilischromosome at the nprE locus (SEQ ID NO:13).

FIG. 30 is a map of p9796-poplar.

FIGS. 31A-B are a nucleotide sequence of p9796-poplar (SEQ ID NO:14).

FIG. 32 is a map of pTrcPoplar.

FIGS. 33A-C are a nucleotide sequence of pTrcPoplar (SEQ ID NO:15).

FIG. 34 is a map of pTrcKudzu yIDI Kan.

FIGS. 35A-C are a nucleotide sequence of pTrcKudzu yIDI Kan (SEQ IDNO:16).

FIG. 36 is a map of pTrcKudzuDXS Kan.

FIGS. 37A-C are a nucleotide sequence of pTrcKudzuDXS Kan (SEQ IDNO:17).

FIG. 38 is a map of pCL PtrcKudzu.

FIGS. 39A-C are a nucleotide sequence of pCL PtrcKudzu (SEQ ID NO:18).

FIG. 40 is a map of pCL PtrcKudzu A3.

FIGS. 41A-C are a nucleotide sequence of pCL PtrcKudzu A3 (SEQ IDNO:19).

FIG. 42 is a map of pCL PtrcKudzu yIDI.

FIGS. 43A-C are a nucleotide sequence of pCL PtrcKudzu yIDI (SEQ IDNO:20).

FIG. 44 is a map of pCL PtrcKudzu DXS.

FIGS. 45A-D are a nucleotide sequence of pCL PtrcKudzu DXS (SEQ IDNO:21).

FIGS. 46A-E show graphs representing isoprene production from biomassfeedstocks. Panel A shows isoprene production from corn stover, Panel Bshows isoprene production from bagasse, Panel C shows isopreneproduction from softwood pulp, Panel D shows isoprene production fromglucose, and Panel E shows isoprene production from cells with noadditional feedstock. Grey squares represent OD₆₀₀ measurements of thecultures at the indicated times post-inoculation and black trianglesrepresent isoprene production at the indicated times post-inoculation.

FIG. 47A shows a graph representing isoprene production by BL21 (λDE3)pTrcKudzu yIDI DXS (kan) in a culture with no glucose added. Squaresrepresent OD₆₀₀, and triangles represent isoprene produced (μg/ml).

FIG. 47B shows a graph representing isoprene production from 1% glucosefeedstock invert sugar by BL21 (λDE3) pTrcKudzu yIDI DXS (kan). Squaresrepresent OD₆₀₀, and triangles represent isoprene produced (μg/ml).

FIG. 47C shows a graph representing isoprene production from 1% invertsugar feedstock by BL21 (λDE3) pTrcKudzu yIDI DXS (kan). Squaresrepresent OD₆₀₀, and triangles represent isoprene produced (μg/ml).

FIG. 47D shows a graph representing isoprene production from 1% AFEXcorn stover feedstock by BL21 (λDE3) pTrcKudzu yIDI DXS (kan). Squaresrepresent OD₆₀₀, and triangles represent isoprene produced (μg/ml).

FIGS. 48A-C show graphs demonstrating the effect of yeast extract ofisoprene production. Panel A shows the time course of optical densitywithin fermentors fed with varying amounts of yeast extract. Panel Bshows the time course of isoprene titer within fermentors fed withvarying amounts of yeast extract. The titer is defined as the amount ofisoprene produced per liter of fermentation broth. Panel C shows theeffect of yeast extract on isoprene production in E. coli grown infed-batch culture.

FIGS. 49A-C show graphs demonstrating isoprene production from a 500 Lbioreactor with E. coli cells containing the pTrcKudzu+yIDI+DXS plasmid.Panel A shows the time course of optical density within the 500-Lbioreactor fed with glucose and yeast extract. Panel B shows the timecourse of isoprene titer within the 500-L bioreactor fed with glucoseand yeast extract. The titer is defined as the amount of isopreneproduced per liter of fermentation broth. Panel C shows the time courseof total isoprene produced from the 500-L bioreactor fed with glucoseand yeast extract.

FIG. 50 is a map of pJMupperpathway2.

FIGS. 51A-C are the nucleotide sequence of pJMupperpathway2 (SEQ IDNO:22).

FIG. 52 is a map of pBS Kudzu #2.

FIG. 53A is a graph showing growth during fermentation time of Bacillusexpressing recombinant kudzu isoprene synthase in 14 liter fed batchfermentation. Black diamonds represent a control strain (BG3594comK)without recombinant isoprene synthase (native isoprene production) andgrey triangles represent CF443, Bacillus strain BG3594comK with pBSKudzu(recombinant isoprene production).

FIG. 53B is a graph showing isoprene production during fermentation timeof Bacillus expressing recombinant kudzu isoprene synthase in 14 literfed batch fermentation. Black diamonds represent a control strain(BG3594comK) without recombinant isoprene synthase (native isopreneproduction) and grey triangles represent CF443, Bacillus strainBG3594comK with pBSKudzu (recombinant isoprene production).

FIG. 54 is a time course of optical density within the 15-L bioreactorfed with glucose.

FIG. 55 is a time course of isoprene titer within the 15-L bioreactorfed with glucose. The titer is defined as the amount of isopreneproduced per liter of fermentation broth.

FIG. 56 is a time course of total isoprene produced from the 15-Lbioreactor fed with glucose.

FIG. 57 is a time course of optical density within the 15-L bioreactorfed with glycerol.

FIG. 58 is a time course of isoprene titer within the 15-L bioreactorfed with glycerol. The titer is defined as the amount of isopreneproduced per liter of fermentation broth.

FIG. 59 is a time course of total isoprene produced from the 15-Lbioreactor fed with glycerol.

FIGS. 60A-60C are the time courses of optical density, mevalonic acidtiter, and specific productivity within the 150-L bioreactor fed withglucose.

FIGS. 61A-61C are the time courses of optical density, mevalonic acidtiter, and specific productivity within the 15-L bioreactor fed withglucose.

FIGS. 62A-62C are the time courses of optical density, mevalonic acidtiter, and specific productivity within the 15-L bioreactor fed withglucose.

FIG. 63A-63C are the time courses of optical density, isoprene titer,and specific productivity within the 15-L bioreactor fed with glucose.

FIGS. 64A-64C are the time courses of optical density, isoprene titer,and specific productivity within the 15-L bioreactor fed with glucose.

FIGS. 65A-65C are the time courses of optical density, isoprene titer,and specific productivity within the 15-L bioreactor fed with glucose.

FIGS. 66A-66C are the time courses of optical density, isoprene titer,and specific productivity within the 15-L bioreactor fed with glucose.

FIG. 67A-67C are the time courses of optical density, isoprene titer,and specific productivity within the 15-L bioreactor fed with glucose.

FIG. 68 is a graph of the calculated adiabatic flame temperatures forSeries A as a function of fuel concentration for various oxygen levels.The figure legend lists the curves in the order in which they appear inthe graph. For example, the first entry in the figure legend (isoprenein air at 40° C.) corresponds to the highest curve in the graph.

FIG. 69 is a graph of the calculated adiabatic flame temperatures forSeries B as a function of fuel concentration for various oxygen levelswith 4% water. The figure legend lists the curves in the order in whichthey appear in the graph.

FIG. 70 is a graph of the calculated adiabatic flame temperatures forSeries C as a function of fuel concentration for various oxygen levelswith 5% CO₂. The figure legend lists the curves in the order in whichthey appear in the graph.

FIG. 71 is a graph of the calculated adiabatic flame temperatures forSeries D as a function of fuel concentration for various oxygen levelswith 10% CO₂. The figure legend lists the curves in the order in whichthey appear in the graph.

FIG. 72 is a graph of the calculated adiabatic flame temperatures forSeries E as a function of fuel concentration for various oxygen levelswith 15% CO₂. The figure legend lists the curves in the order in whichthey appear in the graph.

FIG. 73 is a graph of the calculated adiabatic flame temperatures forSeries F as a function of fuel concentration for various oxygen levelswith 20% CO₂. The figure legend lists the curves in the order in whichthey appear in the graph.

FIG. 74 is a graph of the calculated adiabatic flame temperatures forSeries G as a function of fuel concentration for various oxygen levelswith 30% CO₂. The figure legend lists the curves in the order in whichthey appear in the graph.

FIG. 75A is a table of the conversion of the CAFT Model results fromweight percent to volume percent for series A.

FIG. 75B is a graph of the flammability results from the CAFT model forSeries A in FIG. 68 plotted as volume percent.

FIG. 76A is a table of the conversion of the CAFT Model results fromweight percent to volume percent for series B.

FIG. 76B is a graph of the flammability results from the CAFT model forSeries B in FIG. 69 plotted as volume percent.

FIG. 77 is a figure depicting the flammability test vessel.

FIG. 78A is a graph of the flammability Curve for Test Series 1: 0%Steam, 0 psig, and 40° C.

FIG. 78B is a table summarizing the explosion and non-explosion datapoints for Test Series 1.

FIG. 78C is a graph of the flammability curve for Test Series 1 comparedwith the CAFT Model.

FIG. 79A is a graph of the flammability curve for Test Series 2: 4%Steam, 0 psig, and 40° C.

FIG. 79B is a table summarizing the explosion and non-explosion datapoints for Test Series 2.

FIG. 79C is a graph of the flammability curve for Test Series 2 comparedwith the CAFT Model.

FIGS. 80A-B are a table of the detailed experimental conditions andresults for Test Series 1.

FIG. 81 is a table of the detailed experimental conditions and resultsfor Test Series 2.

FIG. 82 is a graph of the calculated adiabatic flame temperature plottedas a function of fuel concentration for various nitrogen/oxygen ratiosat 3 atmospheres of pressure.

FIG. 83 is a graph of the calculated adiabatic flame temperature plottedas a function of fuel concentration for various nitrogen/oxygen ratiosat 1 atmosphere of pressure.

FIG. 84 is a graph of the flammability envelope constructed using datafrom FIG. 82 and following the methodology described in Example 13. Theexperimental data points (circles) are from tests described herein thatwere conducted at 1 atmosphere initial system pressure.

FIG. 85 is a graph of the flammability envelope constructed using datafrom FIG. 83 and following the methodology described in Example 13. Theexperimental data points (circles) are from tests described herein thatwere conducted at 1 atmosphere initial system pressure.

FIG. 86A is a GC/MS chromatogram of fermentation off-gas.

FIG. 86B is an expansion of FIG. 86A to show minor volatiles present infermentation off-gas.

FIG. 87A is a GC/MS chromatogram of trace volatiles present in off-gasfollowing cryo-trapping at −78° C.

FIG. 87B is a GC/MS chromatogram of trace volatiles present in off-gasfollowing cryo-trapping at −196° C.

FIG. 87C is an expansion of FIG. 87B.

FIG. 87D is an expansion of FIG. 87C.

FIGS. 88A-B are GC/MS chromatogram comparing C5 hydrocarbons frompetroleum-derived isoprene (FIG. 88A) and biologically produced isoprene(FIG. 88B). The standard contains three C5 hydrocarbon impuritieseluting around the main isoprene peak (FIG. 88A). In contrast,biologically produced isoprene contains amounts of ethanol and acetone(run time of 3.41 minutes) (FIG. 88A).

FIG. 89 is a graph of the analysis of fermentation off-gas of an E. coliBL21 (DE3) pTrcIS strain expressing a Kudzu isoprene synthase and fedglucose with 3 g/L yeast extract.

FIG. 90 shows the structures of several impurities that are structurallysimilar to isoprene and may also act as polymerization catalyst poisons.

FIG. 91 is a map of pTrcHis2AUpperPathway (also called pTrcUpperMVA).

FIGS. 92A-92C are the nucleotide sequence of pTrcHis2AUpperPathway (alsocalled pTrcUpperMVA) (SEQ ID NO:23).

FIG. 93 is a time course of optical density within the 15-L bioreactorfed with glucose.

FIG. 94 is a time course of isoprene titer within the 15-L bioreactorfed with glucose. The titer is defined as the amount of isopreneproduced per liter of fermentation broth.

FIG. 95 is a time course of total isoprene produced from the 15-Lbioreactor fed with glucose.

FIG. 96 is a time course of optical density within the 15-L bioreactorfed with invert sugar.

FIG. 97 is a time course of isoprene titer within the 15-L bioreactorfed with invert sugar. The titer is defined as the amount of isopreneproduced per liter of fermentation broth.

FIG. 98 is a time course of total isoprene produced from the 15-Lbioreactor fed with invert sugar.

FIG. 99 is a time course of optical density within the 15-L bioreactorfed with glucose.

FIG. 100 is a time course of isoprene titer within the 15-L bioreactorfed with glucose. The titer is defined as the amount of isopreneproduced per liter of fermentation broth.

FIG. 101 is a time course of isoprene specific activity from the 15-Lbioreactor fed with glucose.

FIG. 102 is a map of pCLPtrcUpperPathwayHGS2.

FIGS. 103A-103C are the nucleotide sequence of pCLPtrcUpperPathwayHGS2(SEQ ID NO:24).

FIG. 104 is a time course of optical density within the 15-L bioreactorfed with glucose.

FIG. 105 is a time course of isoprene titer within the 15-L bioreactorfed with glucose. The titer is defined as the amount of isopreneproduced per liter of fermentation broth.

FIG. 106 is a time course of total isoprene produced from the 15-Lbioreactor fed with glucose.

FIG. 107 is a map of plasmid MCM330 (FRT-cm-FRT-gi1.2-KKDy at attTn7).

FIGS. 108A-108C are the nucleotide sequence of plasmid MCM330 (SEQ IDNO:25).

FIG. 109 is a map of pET24D-Kudzu.

FIGS. 110A-B are the nucleotide sequence of pET24D-Kudzu (SEQ ID NO:26).

FIG. 111A is a time course of optical density within the 15-L bioreactorfed with glucose.

FIG. 111B is a time course of isoprene titer within the 15-L bioreactorfed with glucose. The titer is defined as the amount of isopreneproduced per liter of fermentation broth.

FIG. 111C is a time course of specific productivity of isoprene in the15-L bioreactor fed with glucose.

FIG. 112A is a map of the M. mazei archaeal Lower Pathway operon.

FIGS. 112B-C are the nucleotide sequence of the M. mazei archaeal lowerPathway operon (SEQ ID NO:27).

FIG. 113A is a map of MCM382-pTrcKudzuMVK(mazei).

FIGS. 113B-C are the nucleotide sequence of MCM382-pTrcKudzuMVK(mazei)(SEQ ID NO:28).

FIG. 114A is a map of MCM376-MVK from M. mazei archaeal Lower inpET200D.

FIGS. 114B-C are the nucleotide sequence of MCM376-MVK from M. mazeiarchaeal Lower in pET200D (SEQ ID NO:29).

FIGS. 115A-115D demonstrate that over-expression of MVK and isoprenesynthase results in increased isoprene production. Accumulated isopreneand CO₂ from MCM401 and MCM343 during growth on glucose in 100 mLbioreactors with 100 and 200 μM IPTG induction of isoprene productionwas measured over a 22 hour time course. FIG. 115A is a graph of theaccumulated isoprene (%) from MCM343. FIG. 115B is a graph of theaccumulated isoprene (%) from MCM401. FIG. 115C is a graph of theaccumulated CO₂ (%) from MCM343. FIG. 115D is a graph of the accumulatedCO₂ (%) from MCM401.

FIG. 116 is a time course of optical density within the 15-L bioreactorfed with glucose.

FIG. 117 is a time course of isoprene titer within the 15-L bioreactorfed with glucose. The titer is defined as the amount of isopreneproduced per liter of fermentation broth.

FIG. 118 is a time course of total isoprene produced from the 15-Lbioreactor fed with glucose.

FIG. 119 is a graph of the total carbon dioxide evolution rate (TCER),or metabolic activity profile, within the 15-L bioreactor fed withglucose.

FIG. 120 is a graph of the cell viability during isoprene productionwithin the 15-L bioreactor fed with glucose. TVC/OD is the total viablecounts (colony forming units) in 1 mL of broth per optical density unit(OD₅₅₀).

FIG. 121 is a time course of optical density within the 15-L bioreactorfed with glucose.

FIG. 122 is a time course of isoprene titer within the 15-L bioreactorfed with glucose. The titer is defined as the amount of isopreneproduced per liter of fermentation broth.

FIG. 123 is a time course of total isoprene produced from the 15-Lbioreactor fed with glucose.

FIG. 124 is a time course of volumetric productivity within the 15-Lbioreactor fed with glucose. The volumetric productivity is defined asthe amount of isoprene produced per liter of broth per hour.

FIG. 125 is a time course of instantaneous yield within the 15-Lbioreactor fed with glucose. The instantaneous yield is defined as theamount of isoprene (gram) produced per amount of glucose (gram) fed tothe bioreactor (w/w) during the time interval between the data points.

FIG. 126 is a graph of the total carbon dioxide evolution rate (TCER),or metabolic activity profile, within the 15-L bioreactor fed withglucose.

FIG. 127 is cell viability during isoprene production within the 15-Lbioreactor fed with glucose. TVC/OD is the total viable counts (colonyforming units) in 1 mL of broth per optical density unit (OD₅₅₀).

FIG. 128 is a time course of optical density within the 15-L bioreactorfed with glucose.

FIG. 129 is a time course of isoprene titer within the 15-L bioreactorfed with glucose. The titer is defined as the amount of isopreneproduced per liter of fermentation broth.

FIG. 130 is a time course of total isoprene produced from the 15-Lbioreactor fed with glucose.

FIG. 131 is a graph of total carbon dioxide evolution rate (TCER), ormetabolic activity profile, within the 15-L bioreactor fed with glucose.

FIG. 132 is a graph showing that a transient decrease in the airflow tothe bioreactor caused a spike in the concentration of isoprene in theoff-gas that did not cause a dramatic decrease in metabolic activity(TCER). TCER, or metabolic activity, is the total carbon dioxideevolution rate.

FIG. 133 is a graph of the cell viability during isoprene productionwithin the 15-L bioreactor fed with glucose. TVC/OD is the total viablecounts (colony forming units) in 1 mL of broth per optical density unit(OD₅₅₀).

FIG. 134 is a time course of optical density within the 15-L bioreactorfed with glucose. Dotted vertical lines denote the time interval whenisoprene was introduced into the bioreactor at a rate of 1 g/L/hr.

FIG. 135 is total carbon dioxide evolution rate (TCER), or metabolicactivity profile, within the 15-L bioreactor fed with glucose. Dottedvertical lines denote the time interval when isoprene was introducedinto the bioreactor at a rate of 1 g/L/hr.

FIG. 136 is cell viability during isoprene production within the 15-Lbioreactor fed with glucose. TVC/OD is the total viable counts (colonyforming units) in 1 mL of broth per optical density unit (OD₅₅₀). Dottedvertical lines denote the time interval when isoprene was introducedinto the bioreactor at a rate of 1 g/L/hr.

FIGS. 137A-B are the sequence of Populus alba pET24a: isoprene synthasegene highlighted in bold letters (SEQ ID NO:30).

FIGS. 137C-D are the sequence of Populus nigra pET24a: isoprene synthasegene highlighted in bold letters (SEQ ID NO:31).

FIGS. 137E-F are the sequence of Populus tremuloides pET24a (SEQ IDNO:32).

FIG. 137G is the amino acid sequence of Populus tremuloides isoprenesynthase gene (SEQ ID NO:33).

FIGS. 137H-I are the sequence of Populus trichocarpa pET24a: isoprenesynthase gene highlighted in bold letters (SEQ ID NO:34).

FIGS. 137J-K are the sequence of Populus tremula×Populus alba pET24a:isoprene synthase gene highlighted in bold letters (SEQ ID NO:35).

FIG. 137L is a map of MCM93 which contains the kudzu IspS codingsequence in a pCR2.1 backbone.

FIGS. 137M-N are the sequence of MCM93 (SEQ ID NO:36).

FIG. 137O is a map of pET24D-Kudzu.

FIGS. 137P-Q are the sequence of pET24D-Kudzu (SEQ ID NO:37).

FIG. 138 is isoprene synthase expression data for various poplar speciesas measured in the whole cell head space assay. Y-axis is μg/L/OD ofisoprene produced by 0.2 mL of a culture induced with IPTG.

FIG. 139 is relative activity of Poplar isoprene synthase enzymes asmeasured by DMAPP assay. Poplar enzymes have significantly higheractivity than the isoprene synthase from Kudzu. Poplar [alba×tremula]only had traces (<1%) of activity and is not shown in the plot.

FIG. 140 is a map of pDONR221:19430-hybrid_HGS.

FIG. 141 is the nucleotide sequence of pDONR221:19430-hybrid_HGS, thesequence of Kudzu isoprene synthase codon-optimized for yeast (SEQ IDNO:38).

FIG. 142A is a map of pDW14.

FIGS. 142B-C are the complete nucleotide sequence of pDW14 (SEQ IDNO:39).

FIG. 143 shows induced INVSc-1 strains harboring pDW14 or pYES-DEST52.FIG. 143A. A 4-12% bis tris gel (Novex, Invitrogen) of lysates generatedfrom INVSc-1 strains induced with galactose and stained with SimplyBlueSafeStain (Invitrogen). FIG. 143B. Western blot analysis of the samestrains using the WesternBreeze kit (Invitrogen). Lanes are as follows:1, INVSc-1+pYES-DEST52; 2, INVSc-1+pDW14 (isolate 1); 3, INVSc-1+pDW14(isolate 2). MW (in kDa) is indicated (using the SeeBlue Plus2 molecularweight standard).

FIG. 144 (FIGS. 144A and 144B) shows induced INVSc-1 strains harboringpDW14 or pYES-DEST52. FIG. 144A. OD₆₀₀ of galactose-induced strainsprior to lysis. The y-axis is OD₆₀₀. FIG. 144B. DMAPP assay of isoprenesynthase headspace in control and isoprene synthase-harboring strains.Specific activity was calculated as μg HG/L/OD. Samples are as follows:Control, INVSc-1+pYES-DEST52; HGS-1, INVSc-1+pDW14 (isolate 1); HGS-2,INVSc-1+pDW14 (isolate 2).

FIG. 145A is a map of codon optimized isoprene synthase fluo-opt2v2.

FIG. 145B is the nucleotide sequence of codon optimized isoprenesynthase fluo-opt2v2 (SEQ ID NO:40).

FIG. 146A is a map of pBBR1MCS5.

FIGS. 146B-C are the nucleotide sequence of pBBR1MCS5 (SEQ ID NO:41).

FIG. 147A is a map of pBBR5HGSOpt2_(—)2.

FIGS. 147B-C are the nucleotide sequence of pBBR5HGSOpt2_(—)2 (SEQ IDNO:42).

FIG. 148 is a graph of CER versus fermentation time for strain MCM401,uninduced, induced with IPTG (4×50 tμmol) or IPTG (2×100 tμmol).

FIG. 149 shows concentration of glucose in sugar cane solutions, pHadjusted or not, as a function of the number of autoclaving cycles (onecycle=30 min).

FIG. 150 shows growth curves (OD₆₀₀ as a function of time) ofPseudomonas putida F1 and Pseudomonas fluorescens ATCC13525 on glucose,sugar cane, and inverted sugar cane.

FIG. 151 shows growth curves (OD₆₀₀ as a function of time) of E. coliBL21(DE3), MG1655, ATCC11303 and B REL 606 on glucose, sugar cane, andinverted sugar cane.

FIG. 152 is a map of plasmid pET24 P. alba HGS.

FIG. 153A-B are the nucleotide sequence of plasmid pET24 P. alba HGS(SEQ ID NO:43).

FIG. 154 is a schematic diagram showing restriction sites used forendonuclease digestion to construct plasmid EWL230 and compatiblecohesive ends between BspHI and NcoI sites.

FIG. 155 is a map of plasmid EWL230.

FIGS. 156A-B are the nucleotide sequence of plasmid EWL230 (SEQ IDNO:44).

FIG. 157 is a schematic diagram showing restriction sites used forendonuclease digestion to construct plasmid EWL244 and compatiblecohesive ends between NsiI and PstI sites.

FIG. 158 is a map of plasmid EWL244.

FIGS. 159A-B are the nucleotide sequence of plasmid EWL244 (SEQ IDNO:45).

FIG. 160A is a map of the M. mazei archaeal Lower Pathway operon.

FIGS. 160B-C are the nucleotide sequence of the M. mazei archaeal LowerPathway operon (SEQ ID NO:46).

FIG. 161A is a map of MCM376-MVK from M. mazei archaeal Lower inpET200D.

FIGS. 161B-C are the nucleotide sequence of MCM376-MVK from M. mazeiarchaeal Lower in pET200D (SEQ ID NO:47).

FIG. 162 is a map of plasmid pBBRCMPGI1.5-pgl.

FIGS. 163A-B are the nucleotide sequence of plasmid pBBRCMPGI1.5-pgl(SEQ ID NO:48).

FIGS. 164A-F are graphs of isoprene production by E. coli strainexpressing M. mazei mevalonate kinase, P. alba isoprene synthase, andpgl (RHM111608-2), and grown in fed-batch culture at the 15-L scale.FIG. 164A shows the time course of optical density within the 15-Lbioreactor fed with glucose. FIG. 164B shows the time course of isoprenetiter within the 15-L bioreactor fed with glucose. The titer is definedas the amount of isoprene produced per liter of fermentation broth.Method for calculating isoprene: cumulative isoprene produced in 59 hrs,g/Fermentor volume at 59 hrs, L [=] g/L broth. FIG. 164C also shows thetime course of isoprene titer within the 15-L bioreactor fed withglucose. Method for calculating isoprene: ∫(Instantaneous isopreneproduction rate, g/L/hr)dt from t=0 to 59 hours [=] g/L broth. FIG. 164Dshows the time course of total isoprene produced from the 15-Lbioreactor fed with glucose. FIG. 164E shows volumetric productivitywithin the 15-L bioreactor fed with glucose. FIG. 164F shows carbondioxide evolution rate (CER), or metabolic activity profile, within the15-L bioreactor fed with glucose.

FIGS. 165A-B are graphs showing analysis of off-gas from fermentation in15 L bioreactors. Sample A is strain RM111608-2 sampled at 64.8 hours.Sample B is strain EWL256 was E. coli BL21 (DE3), pCL upper,cmR-gi1.2-yKKDyI, pTrcAlba-mMVK sampled at 34.5 hours. Hydrogen isdetected above the baseline (0.95×10⁻⁸ ton) for both samples.

FIG. 166A shows an exemplary Bioisoprene™ recovery unit.

FIG. 166B shows an exemplary Bioisoprene™ desorption/condensation setup.

FIG. 167 shows a GC/FID chromatogram of a Bioisoprene™ product. Thematerial was determined to be 99.7% pure.

FIG. 168A-C show the GC/FID chromatograms of a Bioisoprene™ samplebefore (A) and after treatment with alumina (B) or silica (C). Theisoprene peak is not shown in these chromatograms.

FIG. 169 shows a diagram of a process and associated apparatus forpurifying isoprene from a fermentation off-gas.

FIG. 170 shows GC/FID chromatogram of partially hydrogenatedBioIsoprene™ monomer. Compound 1 (RT=12.30 min)=3-methyl-1-butene,compound 2 (RT=12.70 min)=2-methylbutane, compound 3 (RT=13.23min)=2-methyl-1-butene, compound 4 (RT=13.53 min)=isoprene, compound 5(RT=14.01 min)=2-methyl-2-butene).

FIG. 171 shows the GC/MS Total Ion Chromatogram for products derved fromthe Amberlyst-15 acid resin-catalyzed dimerization of 2-methyl-2-butene.

FIG. 172 shows the GC/MS Total Ion Chramatogram for products derivedfrom Amberlyst 15 acid resin-catalyzed oligomerization of BioIsoprene™monomer.

FIG. 173 shows a process flow diagram for the conversion of a C5 streaminto a C10/C15 product stream using a dimerization reactor. The C5stream comprises BioIsoprene™ monomer and/or C5 derivatives ofBioIsoprene™ monomer.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides, inter alia, compositions and methods forproducing a fuel constituent from isoprene. Provided herein are fuelconstituents or additives, for example, cyclic isoprene dimers andtrimers, linear isoprene oligomers, aromatic and alicyclic isoprenederivatives, and oxygenated isoprene derivatives. The fuel constituentcan be produced by chemical transformations of a starting materialcomprising a commercially beneficial amount of highly pure isoprene. Inone aspect, the commercially beneficial amount of highly pure isoprenecomprises bioisoprene. In another aspect, a commercially beneficialamount of highly pure isoprene can be bioisoprene. In another aspect, acommercially beneficial amount of highly pure isoprene can be highlypure isoprene compositions produced by culturing cells expressing aheterologous isoprene synthase enzyme. In other aspects, highly pureisoprene undergoes oligomerization to form unsaturated isopreneoligomers such as cyclic dimers or trimers and linear oligomers. Theunsaturated oligomers may be hydrogenated to produce saturatedhydrocarbon fuel constituent. In some embodiment, reaction of highlypure isoprene with alcohols in the presence of an acid catalyst producesfuel oxygenates. In another aspect, the highly pure isoprene ispartially hydrogenated to produce isoamylenes. In some embodiments, anisoamylene product derived from the highly pure isoprene undergoesdimerization to form isodecenes. In some embodiments, isoamyleneproducts derived from the highly pure isoprene react with alcohols inthe presence of an acid catalyst to produce fuel oxygenates.

Bioisoprene derived from renewable carbon can be converted to a varietyof hydrocarbon fuels by chemical catalysis. Provided herein are methodsfor recovering isoprene from fermentation and subsequent conversion tohydrocarbon fuels by chemical catalysis to compounds of higher molecularweight. These methods include, but are not limited to, recovering andpurifying isoprene from fermentation off-gas and subsequent gas orliquid phase catalysis to provide compounds with fuel value. Bothcontinuous and batch mode processes are contemplated within the scope ofthe invention.

As further detailed herein, a bioisoprene composition is distinguishedfrom a petro-isoprene composition in that a bioisoprene composition issubstantially free of any contaminating unsaturated C5 hydrocarbons thatare usually present in petro-isoprene compositions, such as, but notlimited to, 1,3-cyclopentadiene, trans-1,3-pentadiene,cis-1,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne,3-methyl-1-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, andcis-pent-3-ene-1-yne. If any contaminating unsaturated C5 hydrocarbonsare present in the bioisoprene starting material described herein, theyare present in lower levels than that in petro-isoprene compositions.Accordingly, any fuel products derived from bioisoprene compositionsdescribed herein is essentially free of, or contains at lower levelsthan that in fuel products derived from petro-isoprene, anycontaminating unsaturated C5 hydrocarbons or products derived from suchcontaminating unsaturated C5 hydrocarbons. In addition, the sulfurlevels in a bioisoprene composition are lower than the sulfur levels inpetro-isoprene compositions. Fuels products derived from bioisoprenecompositions contain lower levels of sulfur than that in fuel productsderived from petro-isoprene.

Bioisoprene is distinguished from petro-isoprene in that bioisoprene isproduced with other bio-byproducts (compounds derived from thebiological sources and/or associated the biological processes that areobtained together with bioisoprene) that are not present or present inmuch lower levels in petro-isoprene compositions, such as alcohols,aldehydes, ketone and the like. The bio-byproducts may include, but arenot limited to, ethanol, acetone, methanol, acetaldehyde, methacrolein,methyl vinyl ketone, 2-methyl-2-vinyloxirane, cis- andtrans-3-methyl-1,3-pentadiene, a C5 prenyl alcohol (such as3-methyl-3-buten-1-ol or 3-methyl-2-buten-1-ol), 2-heptanone,6-methyl-5-hepten-2-one, 2,4,5-trimethylpyridine,2,3,5-trimethylpyrazine, citronellal, methanethiol, methyl acetate,1-propanol, diacetyl, 2-butanone, 2-methyl-3-buten-2-ol, ethyl acetate,2-methyl-1-propanol, 3-methyl-1-butanal, 3-methyl-2-butanone, 1-butanol,2-pentanone, 3-methyl-1-butanol, ethyl isobutyrate, 3-methyl-2-butenal,butyl acetate, 3-methylbutyl acetate, 3-methyl-3-buten-1-yl acetate,3-methyl-2-buten-1-yl acetate, 3-hexen-1-ol, 3-hexen-1-yl acetate,limonene, geraniol (trans-3,7-dimethyl-2,6-octadien-1-ol), citronellol(3,7-dimethyl-6-octen-1-ol), (E)-3,7-dimethyl-1,3,6-octatriene,(Z)-3,7-dimethyl-1,3,6-octatriene, 2,3-cycloheptenolpyridine, or alinear isoprene polymer (such as a linear isoprene dimer or a linearisoprene trimer derived from the polymerization of multiple isopreneunits). Fuel products derived from bioisoprene contain one or more ofthe bio-byproducts or compounds derived from any of the bio-byproducts.In addition, fuel products derived from bioisoprene may containcompounds formed from these bio-byproducts during subsequent chemicalconversion. Examples of such compounds include those derived fromDiels-Alder cycloaddition of dienophiles to isoprene or fuel derivativesthereof, the oxidation of isoprene or fuel derivatives.

Further, bioisoprene is distinguished from petro-isoprene by carbonfinger-printing. In one aspect, bioisoprene has a higher radioactivecarbon-14 (¹⁴C) content or higher ¹⁴C/¹²C ratio than petro-isoprene.Bioisoprene is produced from renewable carbon sources, thus the ¹⁴Ccontent or the ¹⁴C/¹²C ratio in bioisoprene is the same as that in thepresent atmosphere. Petro-isoprene, on the other hand, is derived fromfossil fuels deposited thousands to millions of years ago, thus the ¹⁴Ccontent or the ¹⁴C/¹²C ratio is diminished due to radioactive decay. Asdiscussed in greater detail herein, the fuel products derived frombioisoprene has higher ¹⁴C content or ¹⁴C/¹²C ratio than fuel productsderived from petro-isoprene. In one embodiment, a fuel product derivedfrom bioisoprene described herein has a ¹⁴C content or ¹⁴C/¹²C ratiosimilar to that in the atmosphere. In another aspect, bioisoprene can beanalytically distinguished from petro-isoprene by the stable carbonisotope ration (¹³C/¹²C), which can be reported as “delta values”represented by the symbol d¹³C. For examples, for isoprene derived fromextractive distillation of C₅ streams from petroleum refineries, δ¹³C isabout −22‰ to about −24‰. This range is typical for light, unsaturatedhydrocarbons derived from petroleum, and products derived frompetroleum-based isoprene typically contain isoprenic units with the sameδ¹³C. Bioisoprene produced by fermentation of corn-derived glucose (δ¹³C−10.73‰) with minimal amounts of other carbon-containing nutrients(e.g., yeast extract) produces isoprene which can be polymerized intopolyisoprene with δ¹³C −14.66‰ to −14.85‰. Products produced from suchbioisoprene are expected to have δ¹³C values that are less negative thanthose derived from petroleum-based isoprene.

Compounds made by these methods include cyclic isoprene dimers andtrimers, linear oligomers, aromatic and alicyclic derivatives.Diisoamylenes are made by methods comprising partial hydrogenation ofbioisoprene compositons. These chemical derivatives of isoprene areuseful as liquid transportation fuels (IsoFuels™) and as fuel additives.

Also provided herein are methods for the production of oxygenatedderivatives of isoprene including alcohols, ketones, esters and ethers.Methods for the synthesis of oxygenated derivatives of isoprene can alsobe performed in liquid or gas phase, using homogeneous and heterogeneouscatalysts. Compounds of this chemical class are also useful as liquidtransportation fuels, and can be used in fuel blends as fuel oxygenatesfor emissions reduction and as fuel modifiers, for example as cetaneboosters for diesel.

While isoprene can be obtained by fractionating petroleum, thepurification of this material is expensive and time-consuming. Petroleumcracking of the C5 stream of hydrocarbons produces only about 15%isoprene. Isoprene is also naturally produced by a variety of microbial,plant, and animal species. In particular, two pathways have beenidentified for the biosynthesis of isoprene: the mevalonate (MVA)pathway and the non-mevalonate (DXP) pathway. Genetically engineeredcell cultures in bioreactors have produced isoprene more efficiently, inlarger quantities, in higher purities and/or with unique impurityprofiles, e.g. as described in U.S. provisional patent application Nos.61/013,386 and 61/013,574, filed on Dec. 13, 2007, WO 2009/076676, U.S.provisional patent application Nos. 61/134,094, 61/134,947, 61/134,011and 61/134,103, filed on Jul. 2, 2008, WO 2010/003007, U.S. provisionalpatent application No. 61/097,163, filed on Sep. 15, 2008, WO2010/031079, U.S. provisional patent application No. 61/097,186, filedon Sep. 15, 2008, WO 2010/031062, U.S. provisional patent applicationNo. 61/097,189, filed on Sep. 15, 2008, WO 2010/031077, U.S. provisionalpatent application No. 61/097,200, filed on Sep. 15, 2008, WO2010/031068, U.S. provisional patent application No. 61/097,204, filedon Sep. 15, 2008, WO 2010/031076, U.S. provisional patent applicationNo. 61/141,652, filed on Dec. 30, 2008, PCT/US09/069,862, U.S. patentapplication Ser. No. 12/335,071, filed Dec. 15, 2008 (US 2009/0203102A1) and U.S. patent application Ser. No. 12/429,143, filed Apr. 23, 2009(US 2010/0003716 A1), which are incorporated by reference in theirentireties.

DEFINITIONS

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention pertains. Although any methodsand materials similar or equivalent to those described herein find usein the practice of the present invention, the preferred methods andmaterials are described herein. Accordingly, the terms definedimmediately below are more fully described by reference to theSpecification as a whole. All documents cited are, in relevant part,incorporated herein by reference. However, the citation of any documentis not to be construed as an admission that it is prior art with respectto the present invention.

As used herein, the singular terms “a,” “an,” and “the” include theplural reference unless the context clearly indicates otherwise.

It is intended that every maximum numerical limitation given throughoutthis specification includes every lower numerical limitation, as if suchlower numerical limitations were expressly written herein. Every minimumnumerical limitation given throughout this specification will includeevery higher numerical limitation, as if such higher numericallimitations were expressly written herein. Every numerical range giventhroughout this specification will include every narrower numericalrange that falls within such broader numerical range, as if suchnarrower numerical ranges were all expressly written herein.

The term “isoprene” refers to 2-methyl-1,3-butadiene (CAS#78-79-5),which is the direct and final volatile C5 hydrocarbon product from theelimination of pyrophosphate from 3,3-dimethylallyl pyrophosphate(DMAPP), and does not involve the linking or polymerization of [an] IPPmolecule(s) to [a] DMAPP molecule(s). The term “isoprene” is notgenerally intended to be limited to its method of production unlessindicated otherwise herein.

As used herein, “biologically produced isoprene” or “bioisoprene” isisoprene produced by any biological means, such as produced bygenetically engineered cell cultures, natural microbials, plants oranimals.

A “bioisoprene composition” refers to a composition that can be producedby any biological means, such as systems (e.g., cells) that areengineered to produce isoprene. It contains isoprene and other compoundsthat are co-produced (including impurities) and/or isolated togetherwith isoprene. A bioisoprene composition usually contains fewerhydrocarbon impurities than isoprene produced from petrochemical sourcesand often requires minimal treatment in order to be of polymerizationgrade. As detailed herein, bioisoprene composition also has a differentimpurity profile from a petrochemically produced isoprene composition.

As used herein, “at least a portion of the isoprene startingcomposition” can refer to at least about 1%, 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%,99.9%, or 100% of the isoprene starting composition undergoing chemicaltransformation.

As used herein, IsoFuels™ refers to fuels including liquidtransportation fuels that are derived from isoprene. BioIsoFuels™ refersto fuels including liquid transportation fuels that are derived frombioisoprene.

The term “oligomerization” as used herein refers to a chemical processfor combining two or more monomer units. “Oligomerization” of isopreneproduces a derivative of isoprene derived from two or more molecules ofisoprene, such as linear dimers of isoprene, cyclic dimers of isoprene,linear trimers of isoprene, cyclic trimers of isoprene and the like.

“Complete hydrogenation”, “Completely hydrogenate” or “Fullyhydrogenate” is defined as the addition of hydrogen (H₂), typically inthe presence of a hydrogenation catalyst, to all unsaturated functionalgroups, such as carbon-carbon double bonds, within a precursor compoundto give fully saturated product compounds. For example, completehydrogenation of isoprene forms isopentane whereby 2 moles of H₂ isconsumed per mole of isoprene.

“Partial hydrogenation” or “Partially hydrogenate” is defined as theaddition of hydrogen (H₂), typically in the presence of a hydrogenationcatalyst, to at least one, but not all unsaturated functional groups,such as carbon-carbon double bonds, within a precursor compound. Theproduct(s) of partial hydrogenation can be further completelyhydrogenated to give fully saturated product compounds. Partialhydrogenation of a diene forms one or more mono-olefins. For example,partial hydrogenation of isoprene can give 3 isomeric isopentenes(2-methylbut-1-ene, 2-methylbut-2-ene and 3-methylbut-1-ene) whereby 1mole of H₂ is consumed per mole of isoprene.

“Selective hydrogenation” or “Selectively hydrogenate” is defined as theaddition of hydrogen (H₂), typically in the presence of a hydrogenationcatalyst, to at least one, but not all unsaturated functional groups,such as carbon-carbon double bonds, within a precursor compound wherebycertain unsaturated functional groups are preferentially hydrogenatedover other unsaturated groups under the chosen conditions. For example,selective hydrogenation of isoprene may form preferentially2-methyl-2-butene, 2-methyl-1-butene, 3-methyl-1-butene or a mixturethereof.

As used herein, the term “polypeptides” includes polypeptides, proteins,peptides, fragments of polypeptides, and fusion polypeptides.

As used herein, an “isolated polypeptide” is not part of a library ofpolypeptides, such as a library of 2, 5, 10, 20, 50 or more differentpolypeptides and is separated from at least one component with which itoccurs in nature. An isolated polypeptide can be obtained, for example,by expression of a recombinant nucleic acid encoding the polypeptide.

By “heterologous polypeptide” is meant a polypeptide whose amino acidsequence is not identical to that of another polypeptide naturallyexpressed in the same host cell. In particular, a heterologouspolypeptide is not identical to a wild-type polypeptide that is found inthe same host cell in nature.

“Codon degeneracy” refers to divergence in the genetic code permittingvariation of the nucleotide sequence without affecting the amino acidsequence of an encoded polypeptide. The skilled artisan is well aware ofthe “codon-bias” exhibited by a specific host cell in usage ofnucleotide codons to specify a given amino acid. Therefore, whensynthesizing a nucleic acid for improved expression in a host cell, itis desirable in some embodiments to design the nucleic acid such thatits frequency of codon usage approaches the frequency of preferred codonusage of the host cell.

As used herein, a “nucleic acid” refers to two or moredeoxyribonucleotides and/or ribonucleotides covalently joined togetherin either single or double-stranded form.

By “recombinant nucleic acid” is meant a nucleic acid of interest thatis free of one or more nucleic acids (e.g., genes) which, in the genomeoccurring in nature of the organism from which the nucleic acid ofinterest is derived, flank the nucleic acid of interest. The termtherefore includes, for example, a recombinant DNA which is incorporatedinto a vector, into an autonomously replicating plasmid or virus, orinto the genomic DNA of a prokaryote or eukaryote, or which exists as aseparate molecule (e.g., a cDNA, a genomic DNA fragment, or a cDNAfragment produced by PCR or restriction endonuclease digestion)independent of other sequences.

By “heterologous nucleic acid” is meant a nucleic acid whose nucleicacid sequence is not identical to that of another nucleic acid naturallyfound in the same host cell. In particular, a heterologous nucleic acidis not identical to a wild-type nucleic acid that is found in the samehost cell in nature.

As used herein, a “vector” means a construct that is capable ofdelivering, and desirably expressing one or more nucleic acids ofinterest in a host cell. Examples of vectors include, but are notlimited to, plasmids, viral vectors, DNA or RNA expression vectors,cosmids, and phage vectors.

As used herein, an “expression control sequence” means a nucleic acidsequence that directs transcription of a nucleic acid of interest. Anexpression control sequence can be a promoter, such as a constitutive oran inducible promoter, or an enhancer. An “inducible promoter” is apromoter that is active under environmental or developmental regulation.The expression control sequence is operably linked to the nucleic acidsegment to be transcribed.

The term “selective marker” or “selectable marker” refers to a nucleicacid capable of expression in a host cell that allows for ease ofselection of those host cells containing an introduced nucleic acid orvector. Examples of selectable markers include, but are not limited to,antibiotic resistance nucleic acids (e.g., kanamycin, ampicillin,carbenicillin, gentamicin, hygromycin, phleomycin, bleomycin, neomycin,or chloramphenicol) and/or nucleic acids that confer a metabolicadvantage, such as a nutritional advantage on the host cell. Exemplarynutritional selective markers include those markers known in the art asamdS, argB, and pyr4.

Compositions and Systems

Isoprene derived from petrochemical sources usually is an impure C5hydrocarbon fraction which requires extensive purification before thematerial is suitable for polymerization or other chemicaltransformations. Several impurities are particularly problematic giventheir structural similarity to isoprene and the fact that they can actas polymerization catalyst poisons. Such compounds include, but are notlimited to, 1,3-cyclopentadiene, cis- and trans-1,3-pentadiene,1,4-pentadiene, 1-pentyne, 2-pentyne, 3-methyl-1-butyne,pent-4-ene-1-yne, trans-pent-3-ene-1-yne, and cis-pent-3-ene-1-yne. Asdetailed below, biologically produced isoprene can be substantially freeof any contaminating unsaturated C5 hydrocarbons without undergoingextensive purification. Some biologically produced isoprene compositionscontain ethanol, acetone, and C5 prenyl alcohols. These components aremore readily removed from the isoprene stream than the isomeric C5hydrocarbon fractions that are present in isoprene compositions derivedfrom petrochemical sources. Further, these impurities can be managed inthe bioprocess, for example by genetic modification of the producingstrain, carbon feedstock, alternative fermentation conditions, recoveryprocess modifications and additional or alternative purificationmethods.

In one aspect, the invention features compositions and systems forproducing a fuel constituent from isoprene comprising: (a) acommercially beneficial amount of highly pure isoprene startingcomposition; and (b) a fuel constituent produced from at least a portionof the highly pure isoprene starting material; where at least a portionof the commercially beneficial amount of highly pure isoprene startingcomposition undergoes a chemical transformation. A highly pure isoprenestarting material is subjected to chemical reactions to produce acommercially beneficial amount of product that is useful for makingfuels. In one aspect, a commercially beneficial amount of highly pureisoprene comprises bioisoprene. In one aspect, a commercially beneficialamount of highly pure isoprene can be bioisoprene.

Exemplary Starting Isoprene Compositions

In some embodiments, the commercially beneficial amount of highly pureisoprene starting composition comprises greater than or about 2, 5, 10,20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800,900, or 1000 mg of isoprene. In some embodiments, the starting isoprenecomposition comprises greater than or about 2, 5, 10, 20, 30, 40, 50,60, 70, 80, 90, 100 g of isoprene. In some embodiments, the startingisoprene composition comprises greater than or about 0.2, 0.5, 1, 2, 5,10, 20, 50, 100, 200, 500, 1000 kg of isoprene. In some embodiments, theamount of isoprene in the starting composition is between about 2 toabout 5,000 mg, such as between about 2 to about 100 mg, about 100 toabout 500 mg, about 500 to about 1,000 mg, about 1,000 to about 2,000mg, or about 2,000 to about 5,000 mg. In some embodiments, the amount ofisoprene in the starting composition is between about 20 to about 5,000mg, about 100 to about 5,000 mg, about 200 to about 2,000 mg, about 200to about 1,000 mg, about 300 to about 1,000 mg, or about 400 to about1,000 mg. In some embodiments, the amount of isoprene in the startingcomposition is between about 2 to about 5,000 g, such as between about 2to about 100 g, about 100 to about 500 g, about 500 to about 1,000 g,about 1,000 to about 2,000 g, or about 2,000 to about 5,000 g. In someembodiments, the amount of isoprene in the starting composition isbetween about 2 to about 5,000 kg, about 10 to about 2,000 kg, about 20to about 1,000 kg, about 20 to about 500 kg, about 30 to about 200 kg,or about 40 to about 100 kg. In some embodiments, greater than or about20, 25, 30, 40, 50, 60, 70, 80, 90, or 95% (w/w) of the volatile organicfraction of the starting composition is isoprene.

In some embodiments, the highly pure isoprene starting compositioncomprises greater than or about 98.0, 98.5, 99.0, 99.5, or 100% isopreneby weight compared to the total weight of all C5 hydrocarbons in thestarting composition. In some embodiments, the highly pure isoprenestarting composition comprises greater than or about 99.90, 99.92,99.94, 99.96, 99.98, or 100% isoprene by weight compared to the totalweight of all C5 hydrocarbons in the starting composition. In someembodiments, the starting composition has a relative detector responseof greater than or about 98.0, 98.5, 99.0, 99.5, or 100% for isoprenecompared to the detector response for all C5 hydrocarbons in thestarting composition. In some embodiments, the starting composition hasa relative detector response of greater than or about 99.90, 99.91,99.92, 99.93, 99.94, 99.95, 99.96, 99.97, 99.98, 99.99, or 100% forisoprene compared to the detector response for all C5 hydrocarbons inthe starting composition. In some embodiments, the starting isoprenecomposition comprises between about 98.0 to about 98.5, about 98.5 toabout 99.0, about 99.0 to about 99.5, about 99.5 to about 99.8, about99.8 to 100% isoprene by weight compared to the total weight of all C5hydrocarbons in the starting composition. In some embodiments, thestarting isoprene composition comprises between about 99.90 to about99.92, about 99.92 to about 99.94, about 99.94 to about 99.96, about99.96 to about 99.98, about 99.98 to 100% isoprene by weight compared tothe total weight of all C5 hydrocarbons in the starting composition.

In some embodiments, the highly pure isoprene starting compositioncomprises less than or about 2.0, 1.5, 1.0, 0.5, 0.2, 0.12, 0.10, 0.08,0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or0.00001% C5 hydrocarbons other than isoprene (such 1,3-cyclopentadiene,cis-1,3-pentadiene, trans-1,3-pentadiene, 1,4-pentadiene, 1-pentyne,2-pentyne, 1-pentene, 2-methyl-1-butene, 3-methyl-1-butyne,pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne) byweight compared to the total weight of all C5 hydrocarbons in thestarting composition. In some embodiments, the starting composition hasa relative detector response of less than or about 2.0, 1.5, 1.0, 0.5,0.2, 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005,0.0001, 0.00005, or 0.00001% for C5 hydrocarbons other than isoprenecompared to the detector response for all C5 hydrocarbons in thestarting composition. In some embodiments, the starting composition hasa relative detector response of less than or about 2.0, 1.5, 1.0, 0.5,0.2, 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005,0.0001, 0.00005, or 0.00001% for 1,3-cyclopentadiene,cis-1,3-pentadiene, trans-1,3-pentadiene, 1,4-pentadiene, 1-pentyne,2-pentyne, 1-pentene, 2-methyl-1-butene, 3-methyl-1-butyne,pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-ynecompared to the detector response for all C5 hydrocarbons in thestarting composition. In some embodiments, the highly pure isoprenestarting composition comprises between about 0.02 to about 0.04%, about0.04 to about 0.06%, about 0.06 to 0.08%, about 0.08 to 0.10%, or about0.10 to about 0.12% C5 hydrocarbons other than isoprene (such1,3-cyclopentadiene, cis-1,3-pentadiene, trans-1,3-pentadiene,1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene, 2-methyl-1-butene,3-methyl-1-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, orcis-pent-3-ene-1-yne) by weight compared to the total weight of all C5hydrocarbons in the starting composition.

In some embodiments, the highly pure isoprene starting compositioncomprises less than or about 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05,0.01, or 0.005 μg/L of a compound that inhibits the polymerization ofisoprene for any compound in the starting composition that inhibits thepolymerization of isoprene. In some embodiments, the starting isoprenecomposition comprises between about 0.005 to about 50, such as about0.01 to about 10, about 0.01 to about 5, about 0.01 to about 1, about0.01 to about 0.5, or about 0.01 to about 0.005 μg/L of a compound thatinhibits the polymerization of isoprene for any compound in the startingcomposition that inhibits the polymerization of isoprene. In someembodiments, the starting isoprene composition comprises less than orabout 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 μg/L of ahydrocarbon other than isoprene (such 1,3-cyclopentadiene,cis-1,3-pentadiene, trans-1,3-pentadiene, 1,4-pentadiene, 1-pentyne,2-pentyne, 1-pentene, 2-methyl-1-butene, 3-methyl-1-butyne,pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne). Insome embodiments, the starting isoprene composition comprises betweenabout 0.005 to about 50, such as about 0.01 to about 10, about 0.01 toabout 5, about 0.01 to about 1, about 0.01 to about 0.5, or about 0.01to about 0.005 μg/L of a hydrocarbon other than isoprene. In someembodiments, the starting isoprene composition comprises less than orabout 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 μg/L of aprotein or fatty acid (such as a protein or fatty acid that is naturallyassociated with natural rubber).

In some embodiments, the highly pure isoprene starting compositioncomprises less than or about 10, 5, 1, 0.8, 0.5, 0.1, 0.05, 0.01, or0.005 ppm of alpha acetylenes, piperylenes, acetonitrile, or1,3-cyclopentadiene. In some embodiments, the starting isoprenecomposition comprises less than or about 5, 1, 0.5, 0.1, 0.05, 0.01, or0.005 ppm of sulfur or allenes. In some embodiments, the startingisoprene composition comprises less than or about 30, 20, 15, 10, 5, 1,0.5, 0.1, 0.05, 0.01, or 0.005 ppm of all acetylenes (such as 1-pentyne,2-pentyne, 3-methyl-1-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne,and cis-pent-3-ene-1-yne). In some embodiments, the starting isoprenecomposition comprises less than or about 2000, 1000, 500, 200, 100, 50,40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 ppm of isoprenedimers, such as cyclic isoprene dimers (e.g., cyclic C10 compoundsderived from the dimerization of two isoprene units).

In some embodiments, the highly pure isoprene starting compositionincludes ethanol, acetone, methanol, acetaldehyde, methacrolein, methylvinyl ketone, 2-methyl-2-vinyloxirane, cis- andtrans-3-methyl-1,3-pentadiene, a C5 prenyl alcohol (such as3-methyl-3-buten-1-ol or 3-methyl-2-buten-1-ol), or any two or more ofthe foregoing. In particular embodiments, the starting isoprenecomposition comprises greater than or about 0.005, 0.01, 0.05, 0.1, 0.5,1, 5, 10, 20, 30, 40, 60, 80, 100, or 120 μg/L of ethanol, acetone,methanol, acetaldehyde, methacrolein, methyl vinyl ketone,2-methyl-2-vinyloxirane, cis- and trans-3-methyl-1,3-pentadiene, a C5prenyl alcohol (such as 3-methyl-3-buten-1-ol or 3-methyl-2-buten-1-ol),or any two or more of the foregoing. In some embodiments, the isoprenecomposition comprises between about 0.005 to about 120, such as about0.01 to about 80, about 0.01 to about 60, about 0.01 to about 40, about0.01 to about 30, about 0.01 to about 20, about 0.01 to about 10, about0.1 to about 80, about 0.1 to about 60, about 0.1 to about 40, about 5to about 80, about 5 to about 60, or about 5 to about 40 μg/L ofethanol, acetone, methanol, acetaldehyde, methacrolein, methyl vinylketone, 2-methyl-2-vinyloxirane, cis- and trans-3-methyl-1,3-pentadiene,a C5 prenyl alcohol, or any two or more of the foregoing.

In some embodiments, the highly pure isoprene starting compositionincludes one or more of the following components: 2-heptanone,6-methyl-5-hepten-2-one, 2,4,5-trimethylpyridine,2,3,5-trimethylpyrazine, citronellal, acetaldehyde, methanethiol, methylacetate, 1-propanol, diacetyl, 2-butanone, 2-methyl-3-buten-2-ol, ethylacetate, 2-methyl-1-propanol, 3-methyl-1-butanal, 3-methyl-2-butanone,1-butanol, 2-pentanone, 3-methyl-1-butanol, ethyl isobutyrate,3-methyl-2-butenal, butyl acetate, 3-methylbutyl acetate,3-methyl-3-buten-1-yl acetate, 3-methyl-2-buten-1-yl acetate,3-hexen-1-ol, 3-hexen-1-yl acetate, limonene, geraniol(trans-3,7-dimethyl-2,6-octadien-1-ol), citronellol(3,7-dimethyl-6-octen-1-ol), (E)-3,7-dimethyl-1,3,6-octatriene,(Z)-3,7-dimethyl-1,3,6-octatriene, 2,3-cycloheptenolpyridine, or alinear isoprene polymer (such as a linear isoprene dimer or a linearisoprene trimer derived from the polymerization of multiple isopreneunits). In various embodiments, the amount of one of these componentsrelative to amount of isoprene in units of percentage by weight (i.e.,weight of the component divided by the weight of isoprene times 100) isgreater than or about 0.01, 0.02, 0.05, 0.1, 0.5, 1, 5, 10, 20, 30, 40,50, 60, 70, 80, 90, 100, or 110% (w/w). In some embodiments, therelative detector response for the second compound compared to thedetector response for isoprene is greater than or about 0.01, 0.02,0.05, 0.1, 0.5, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 110%.In various embodiments, the amount of one of these components relativeto amount of isoprene in units of percentage by weight (i.e., weight ofthe component divided by the weight of isoprene times 100) is betweenabout 0.01 to about 105% (w/w), such as about 0.01 to about 90, about0.01 to about 80, about 0.01 to about 50, about 0.01 to about 20, about0.01 to about 10, about 0.02 to about 50, about 0.05 to about 50, about0.1 to about 50, or 0.1 to about 20% (w/w).

In some embodiments, at least a portion of the highly pure isoprenestarting composition is in a gas phase. In some embodiments, at least aportion of the highly pure isoprene starting composition is in a liquidphase (such as a condensate). In some embodiments, at least a portion ofthe highly pure isoprene starting composition is in a solid phase. Insome embodiments, at least a portion of the highly pure isoprenestarting composition is absorbed to a solid support, such as a supportthat includes silica and/or activated carbon. In some embodiments, thestarting isoprene composition is mixed with one or more solvents. Insome embodiments, the starting isoprene composition is mixed with one ormore gases.

In other embodiments, the commercially beneficial amount of highly pureisoprene starting composition is produced by a biological process. Insome preferred embodiments, the highly pure isoprene startingcomposition is a bioisoprene composition produced by culturing cellsthat produce greater than about 400 nmole of isoprene/gram of cells forthe wet weight of the cells/hour (nmole/g_(wcm)/hr) of isoprene. In oneembodiment, the bioisoprene composition is produced by culturing cellsthat convert more than about 0.002% of the carbon in a cell culturemedium into isoprene. In other embodiments, the cells have aheterologous nucleic acid that (i) encodes an isoprene synthasepolypeptide, e.g. a naturally-occurring polypeptide from a plant such asPueraria, and (ii) is operably linked to a promoter, e.g. a T7 promoter.Other isoprene synthase polypeptides, for example, from poplar andvariants of naturally-occurring as well as parent isoprene synthase, canbe used to produce bioisoprene. Examples of isoprene synthase and itsvariants that can be used are described in U.S. application Ser. No.12/429,143, which is incorporated herein in its entirety.

In some embodiments, the cells are cultured in a culture medium thatincludes a carbon source, such as, but not limited to, a carbohydrate,glycerol, glycerine, dihydroxyacetone, one-carbon source, oil, animalfat, animal oil, fatty acid, lipid, phospholipid, glycerolipid,monoglyceride, diglyceride, triglyceride, renewable carbon source,polypeptide (e.g., a microbial or plant protein or peptide), yeastextract, component from a yeast extract, or any combination of two ormore of the foregoing. In some embodiments, the cells are cultured underlimited glucose conditions. In some embodiment, the cells furthercomprise a heterologous nucleic acid encoding an IDI polypeptide. Insome embodiments, the cells further comprise a heterologous nucleic acidencoding an MDV pathway polypeptide. In some embodiment, the startingisoprene composition is an isoprene composition as descried or isproduced by culturing any of the cells described in U.S. provisionalpatent application Nos. 61/134,094, filed on Jul. 2, 2008, WO2010/003007, and U.S. patent application Ser. No. 12/335,071, filed Dec.15, 2008 (US 2009/0203102 A1), which are incorporated by reference intheir entireties.

In some embodiments, the highly pure isoprene starting compositioncomprises a gas phase (off-gas) produced by cells in culture thatproduces isoprene. In some embodiments, the gas phase has a nonflammableconcentration of isoprene. In some embodiments, the gas phase comprisesless than about 9.5% (volume) oxygen. In some embodiments, the gas phasecomprises greater than or about 9.5% (volume) oxygen, and theconcentration of isoprene in the gas phase is less than the lowerflammability limit or greater than the upper flammability limit. In someembodiments, the portion of the gas phase other than isoprene comprisesbetween about 0% to about 100% (volume) oxygen, such as between about10% to about 100% (volume) oxygen. In some embodiments, the portion ofthe gas phase other than isoprene comprises between about 0% to about99% (volume) nitrogen. In some embodiments, the portion of the gas phaseother than isoprene comprises between about 1% to about 50% (volume)CO₂.

In some embodiments, the highly pure isoprene starting compositionincludes one or more of the following: an alcohol, an aldehyde, aketone, or an ester (such as any of the alcohols, aldehydes, ketones oresters described herein). In some embodiments, the isoprene compositionincludes (i) an alcohol and an aldehyde, (ii) an alcohol and a ketone,(iii) an aldehyde and a ketone, or (iv) an alcohol, an aldehyde, and aketone. In some embodiments, any of the isoprene compositions furtherincludes an ester.

In some embodiments, the highly pure isoprene starting compositionderived from a biological source (such as a cell culture) contains oneor more of the following: methanol, acetaldehyde, ethanol, methanethiol,1-butanol, 3-methyl-1-propanol, acetone, acetic acid, 2-butanone,2-methyl-1-butanol, or indole. In some embodiments, the startingisoprene composition contains 1 ppm or more of one or more of thefollowing: methanol, acetaldehyde, ethanol, methanethiol, 1-butanol,3-methyl-1-propanol, acetone, acetic acid, 2-butanone,2-methyl-1-butanol, or indole. In some embodiments, the concentration ofmore of one or more of the following: methanol, acetaldehyde, ethanol,methanethiol, 1-butanol, 3-methyl-1-propanol, acetone, acetic acid,2-butanone, 2-methyl-1-butanol, or indole, is between about 1 to about10,000 ppm in a starting isoprene composition (such as off-gas before itis purified). In some embodiments, the starting isoprene composition(such as off-gas after it has undergone one or more purification steps)includes one or more of the following: methanol, acetaldehyde, ethanol,methanethiol, 1-butanol, 3-methyl-1-propanol, acetone, acetic acid,2-butanone, 2-methyl-1-butanol, or indole, at a concentration betweenabout 1 to about 100 ppm, such as about 1 to about 10 ppm, about 10 toabout 20 ppm, about 20 to about 30 ppm, about 30 to about 40 ppm, about40 to about 50 ppm, about 50 to about 60 ppm, about 60 to about 70 ppm,about 70 to about 80 ppm, about 80 to about 90 ppm, or about 90 to about100 ppm. In some embodiments, the starting isoprene composition containsless than 1 ppm of methanethiol (a potent catalyst poison and a sourceof sulfur in the final fuel product) Volatile organic compounds fromcell cultures (such as volatile organic compounds in the headspace ofcell cultures) can be analyzed using standard methods such as thosedescribed herein or other standard methods such as proton transferreaction-mass spectrometry (see, for example, Bunge et al., Applied andEnvironmental Microbiology, 74(7):2179-2186, 2008 which is herebyincorporated by reference in its entirety, particular with respect tothe analysis of volatile organic compounds).

The invention also contemplates the use of highly pure isoprene startingcomposition that is derived from a biological source (such as a cellculture) the co-produces isoprene and hydrogen. In some embodiments, thestarting bioisoprene compositions comprise isoprene and hydrogen inratios ranging from at least one molar percent of isoprene for everythree molar percent of hydrogen to at least one molar percent ofisoprene for every four molar percent of hydrogen. In some embodiments,the starting bioisoprene compositions comprise isoprene and hydrogen inmolar ratios of about 1 to 9, 2 to 8, 3 to 7, 4 to 6, 5 to 5, 6 to 4, 7to 3, 8 to 2, or 9 to 1. In some embodiments, the composition furthercomprises from 1 to 11 molar percent isoprene and from 4 to 44 molarpercent hydrogen. In some embodiments, the composition further comprisesoxygen, carbon dioxide, or nitrogen. In some embodiments, thecomposition further comprises from 0 to 21 molar percent oxygen, from 18to 44 molar percent carbon dioxide, and from 0 to 78 molar percentnitrogen. In some embodiments, the composition further comprises1.0×10⁻⁴ molar percent or less of non-methane volatile impurities. Insome embodiments, the non-methane volatile impurities comprise one ormore of the following: 2-heptanone, 6-methyl-5-hepten-2-one,2,4,5-trimethylpyridine, 2,3,5-trimethylpyrazine, citronellal,acetaldehyde, methanethiol, methyl acetate, 1-propanol, diacetyl,2-butanone, 2-methyl-3-buten-2-ol, ethyl acetate, 2-methyl-1-propanol,3-methyl-1-butanal, 3-methyl-2-butanone, 1-butanol, 2-pentanone,3-methyl-1-butanol, ethyl isobutyrate, 3-methyl-2-butenal, butylacetate, 3-methylbutyl acetate, 3-methyl-3-buten-1-yl acetate,3-methyl-2-buten-1-yl acetate, 3-hexen-1-ol, 3-hexen-1-yl acetate,limonene, geraniol (trans-3,7-dimethyl-2,6-octadien-1-ol), citronellol(3,7-dimethyl-6-octen-1-ol), (E)-3,7-dimethyl-1,3,6-octatriene,(Z)-3,7-dimethyl-1,3,6-octatriene, 2,3-cycloheptenolpyridine, or alinear isoprene polymer (such as a linear isoprene dimer or a linearisoprene trimer derived from the polymerization of multiple isopreneunits). In some embodiments, the non-methane volatile impuritiescomprise one or more of the following: the isoprene composition includesone or more of the following: an alcohol, an aldehyde, or a ketone (suchas any of the alcohols, aldehydes, or ketones described herein). In someembodiments, the isoprene composition includes (i) an alcohol and analdehyde, (ii) an alcohol and a ketone, (iii) an aldehyde and a ketone,or (iv) an alcohol, an aldehyde, and a ketone. In some embodiments, thenon-methane volatile impurities comprise one or more of the following:methanol, acetaldehyde, ethanol, methanethiol, 1-butanol,3-methyl-1-propanol, acetone, acetic acid, 2-butanone,2-methyl-1-butanol, or indole.

Techniques for producing isoprene in cultures of cells that produceisoprene are described in U.S. provisional patent application Nos.61/013,386 and 61/013,574, filed on Dec. 13, 2007, WO 2009/076676, U.S.provisional patent application Nos. 61/134,094, 61/134,947, 61/134,011and 61/134,103, filed on Jul. 2, 2008, WO 2010/003007, U.S. provisionalpatent application No. 61/097,163, filed on Sep. 15, 2008, WO2010/031079, U.S. provisional patent application No. 61/097,186, filedon Sep. 15, 2008, WO 2010/031062, U.S. provisional patent applicationNo. 61/097,189, filed on Sep. 15, 2008, WO 2010/031077, U.S. provisionalpatent application No. 61/097,200, filed on Sep. 15, 2008, WO2010/031068, U.S. provisional patent application No. 61/097,204, filedon Sep. 15, 2008, WO 2010/031076, U.S. provisional patent applicationNo. 61/141,652, filed on Dec. 30, 2008, PCT/US09/069,862, U.S. patentapplication Ser. No. 12/335,071, filed Dec. 15, 2008 (US 2009/0203102A1) and U.S. patent application Ser. No. 12/429,143, filed Apr. 23, 2009(US 2010/0003716 A1), the teachings of which are incorporated herein byreference for the purpose of teaching techniques for producing andrecovering isoprene by such a process. In any case, U.S. provisionalpatent application Nos. 61/013,386 and 61/013,574, filed on Dec. 13,2007, WO 2009/076676, U.S. provisional patent application Nos.61/134,094, 61/134,947, 61/134,011 and 61/134,103, filed on Jul. 2,2008, WO 2010/003007, U.S. provisional patent application No.61/097,163, filed on Sep. 15, 2008, WO 2010/031079, U.S. provisionalpatent application No. 61/097,186, filed on Sep. 15, 2008, WO2010/031062, U.S. provisional patent application No. 61/097,189, filedon Sep. 15, 2008, WO 2010/031077, U.S. provisional patent applicationNo. 61/097,200, filed on Sep. 15, 2008, WO 2010/031068, U.S. provisionalpatent application No. 61/097,204, filed on Sep. 15, 2008, WO2010/031076, U.S. provisional patent application No. 61/141,652, filedon Dec. 30, 2008, PCT/US09/069,862, U.S. patent application Ser. No.12/335,071, filed Dec. 15, 2008 (US 2009/0203102 A1) and U.S. patentapplication Ser. No. 12/429,143, filed Apr. 23, 2009 (US 2010/0003716A1) teach compositions and methods for the production of increasedamounts of isoprene in cell cultures. U.S. patent application Ser. No.12/335,071, filed Dec. 15, 2008 and US 2009/0203102 A1 further teachescompositions and methods for co-production of isoprene and hydrogen fromcultured cells. In particular, these compositions and methodscompositions and methods increase the rate of isoprene production andincrease the total amount of isoprene that is produced. For example,cell culture systems that generate 4.8×10⁴ nmole/g_(wcm)/hr of isoprenehave been produced (Table 1). The efficiency of these systems isdemonstrated by the conversion of about 2.2% of the carbon that thecells consume from a cell culture medium into isoprene. As shown in theExamples and Table 2, approximately 3 g of isoprene per liter of brothwas generated. If desired, even greater amounts of isoprene can beobtained using other conditions, such as those described herein. In someembodiments, a renewable carbon source is used for the production ofisoprene. In some embodiments, the production of isoprene is decoupledfrom the growth of the cells. In some embodiments, the concentrations ofisoprene and any oxidants are within the nonflammable ranges to reduceor eliminate the risk that a fire may occur during production orrecovery of isoprene. The compositions and methods are desirable becausethey allow high isoprene yield per cell, high carbon yield, highisoprene purity, high productivity, low energy usage, low productioncost and investment, and minimal side reactions. This efficient, largescale, biosynthetic process for isoprene production provides an isoprenesource for synthetic isoprene-based products such as rubber and providesa desirable, low-cost alternative to using natural rubber.

As discussed further below, the amount of isoprene produced by cells canbe greatly increased by introducing a heterologous nucleic acid encodingan isoprene synthase polypeptide (e.g., a plant isoprene synthasepolypeptide) into the cells. Isoprene synthase polypeptides convertdimethyl allyl diphosphate (DMAPP) into isoprene. As shown in theExamples, a heterologous Pueraria Montana (kudzu) isoprene synthasepolypeptide was expressed in a variety of host cells, such asEscherichia coli, Panteoa citrea, Bacillus subtilis, Yarrowialipolytica, and Trichoderma reesei. All of these cells produced moreisoprene than the corresponding cells without the heterologous isoprenesynthase polypeptide. As illustrated in Tables 1 and 2, large amounts ofisoprene are produced using the methods described herein. For example,B. subtilis cells with a heterologous isoprene synthase nucleic acidproduced approximately 10-fold more isoprene in a 14 liter fermentorthan the corresponding control B. subtilis cells without theheterologous nucleic acid (Table 2). The production of 300 mg ofisoprene per liter of broth (mg/L, wherein the volume of broth includesboth the volume of the cell medium and the volume of the cells) by E.coli and 30 mg/L by B. subtilis in fermentors indicates that significantamounts of isoprene can be generated (Table 2). If desired, isoprene canbe produced on an even larger scale or other conditions described hereincan be used to further increase the amount of isoprene. The vectorslisted in Tables 1 and 2 and the experimental conditions are describedin further detail below and in the Examples section.

TABLE 1 Exemplary yields of isoprene from a shake flask using the cellcultures and methods described herein. The assay for measuring isopreneproduction is described in Example I, part II. For this assay, a samplewas removed at one or more time points from the shake flask and culturedfor 30 minutes. The amount of isoprene produced in this sample was thenmeasured. The headspace concentration and specific rate of isopreneproduction are listed in Table 1 and described further herein. IsopreneProduction in a Headspace vial* Headspace Specific Rate concentrationμg/L_(broth)/hr/OD Strain μg/L_(gas) (nmol/g_(wcm)/hr) E. coliBL21/pTrcKudzu IS 1.40 53.2 (781.2) E. coli BL21/pCL DXS yidi 7.61 289.1(4.25 × 10³) Kudzu IS E. coli BL21/MCM127 23.0 874.1 (1.28 × 10⁴) withkudzu IS and entire MVA pathway E. coli BL21/pET 1.49 56.6 (831.1)N-HisKudzu IS Pantoea citrea/pTrcKudzu IS 0.66 25.1 (368.6) E. coli w/Poplar IS — 5.6 (82.2) [Miller (2001)] Bacillis licheniformis Fall — 4.2(61.4) U.S. Pat. No. 5,849,970 Yarrowia lipolytica with ~0.05 μg/L ~2(~30) kudzu isoprene synthase Trichoderma reesei with ~0.05 μg/L ~2(~30) kudzu isoprene synthase E. coli BL21/pTrcKKD_(y)I_(k)IS 85.9 3.2 ×10³ (4.8 × 10⁴) with kudzu IS and lower MVA pathway *Normalized to 1 mLof 1 OD₆₀₀, cultured for 1 hour in a sealed headspace vial with a liquidto headspace volume ratio of 1:19.

TABLE 2 Exemplary yields of isoprene in a fermentor using the cellcultures and methods described herein. The assay for measuring isopreneproduction is described in Example I, part II. For this assay, a sampleof the off-gas of the fermentor was taken and analyzed for the amount ofisoprene. The peak headspace concentration (which is the highestheadspace concentration during the fermentation), titer (which is thecumulative, total amount of isoprene produced per liter of broth), andpeak specific rate of isoprene production (which is the highest specificrate during the fermentation) are listed in Table 2 and describedfurther herein. Isoprene Production in Fermentors Peak Peak HeadspaceSpecific rate concentration** Titer μg/L_(broth)/hr/OD Strain(μg/L_(gas)) (mg/L_(broth)) (nmol/g_(wcm)/hr) E. coli BL21/pTrcKudzu 5241.2 37  with Kudzu IS (543.3) E. coli FM5/pTrcKudzu 3 3.5  21.4 IS(308.1) E. coli BL21/triple strain 285 300 240   (DXS, yidi, IS) (3.52 ×10³) E. coli FM5/triple strain 50.8 29 180.8 (DXS, yidi, IS) (2.65 ×10³) E. coli/MCM127 with 3815 3044 992.5 Kudzu IS and entire (1.46 ×10⁴) MVA pathway E. coli BL21/pCLPtrc 2418 1640 1248   Upper Pathwaygi1.2 (1.83 × 10⁴) integrated lower pathway pTrcKudzu E. coliBL21/MCM401 13991 23805 3733   with 4 × 50 μM IPTG (5.49 × 10⁴) E. coliBL21/MCM401 22375 19541 5839.5  with 2 × 1000 μM IPTG (8.59 × 10⁴) E.coli BL21/pCLPtrc 3500 3300 1088   UpperPathwayHGS2- (1.60 × 10⁴)pTrcKKDyIkIS Bacillus subtilis 1.5 2.5  0.8 wild-type  (11.7) BacilluspBS Kudzu IS 16.6 ~30  5 (over  (73.4) 100 hrs) Bacillus Marburg 60512.04 0.61  24.5 [Wagner and Fall (1999)] (359.8) Bacillus Marburg 60510.7 0.15  6.8 Fall U.S. Pat. No. (100)   5,849,970 E. coli 2.03 × 10⁴3.22 × 10⁴  5.9 × 10³ BL21/pCLPtrcUpperPath (8.66 × 10⁴) way andgil.2KKDyI and pTrcAlba-mMVK E. coli 3.22 × 10⁴ 6.05 × 10⁴ 1.28 × 10⁴BL21/pCLPtrcUpper (1.88 × 10⁵) Pathway and gi1.2KKDyI and pTrcAlba-mMVKplus pBBRCMPGI1.5pgl **Normalized to an off-gas flow rate of 1 vvm (1volume off-gas per 1 L_(broth) per minute).

Additionally, isoprene production by cells that contain a heterologousisoprene synthase nucleic acid can be enhanced by increasing the amountof a 1-deoxy-D-xylulose-5-phosphate synthase (DXS) polypeptide and/or anisopentenyl diphosphate isomerase (IDI) polypeptide expressed by thecells. For example, a DXS nucleic acid and/or an IDI nucleic acid can beintroduced into the cells. The DXS nucleic acid may be a heterologousnucleic acid or a duplicate copy of an endogenous nucleic acid.Similarly, the IDI nucleic acid may be a heterologous nucleic acid or aduplicate copy of an endogenous nucleic acid. In some embodiments, theamount of DXS and/or IDI polypeptide is increased by replacing theendogenous DXS and/or IDI promoters or regulatory regions with otherpromoters and/or regulatory regions that result in greater transcriptionof the DXS and/or IDI nucleic acids. In some embodiments, the cellscontain both a heterologous nucleic acid encoding an isoprene synthasepolypeptide (e.g., a plant isoprene synthase nucleic acid) and aduplicate copy of an endogenous nucleic acid encoding an isoprenesynthase polypeptide.

The encoded DXS and IDI polypeptides are part of the DXP pathway for thebiosynthesis of isoprene (FIG. 19A). DXS polypeptides convert pyruvateand D-glyceraldehyde-3-phosphate into 1-deoxy-D-xylulose-5-phosphate.While not intending to be bound by any particular theory, it is believedthat increasing the amount of DXS polypeptide increases the flow ofcarbon through the DXP pathway, leading to greater isoprene production.IDI polypeptides catalyze the interconversion of isopentenyl diphosphate(IPP) and dimethyl allyl diphosphate (DMAPP). While not intending to bebound by any particular theory, it is believed that increasing theamount of IDI polypeptide in cells increases the amount (and conversionrate) of IPP that is converted into DMAPP, which in turn is convertedinto isoprene.

For example, fermentation of E. coli cells with a kudzu isoprenesynthase, S. cerevisia IDI, and E. coli DXS nucleic acids was used toproduce isoprene. The levels of isoprene varied from 50 to 300 μg/L overa time period of 15 hours (Example 7, part VII).

In some embodiments, the presence of heterologous or extra endogenousisoprene synthase, IDI, and DXS nucleic acids causes cells to grow morereproducibly or remain viable for longer compared to the correspondingcell with only one or two of these heterologous or extra endogenousnucleic acids. For example, cells containing heterologous isoprenesynthase, IDI, and DXS nucleic acids grew better than cells with onlyheterologous isoprene synthase and DXS nucleic acids or with only aheterologous isoprene synthase nucleic acid. Also, heterologous isoprenesynthase, IDI, and DXS nucleic acids were successfully operably linkedto a strong promoter on a high copy plasmid that was maintained by E.coli cells, suggesting that large amounts of these polypeptides could beexpressed in the cells without causing an excessive amount of toxicityto the cells. While not intending to be bound to a particular theory, itis believed that the presence of heterologous or extra endogenousisoprene synthase and IDI nucleic acids may reduce the amount of one ormore potentially toxic intermediates that would otherwise accumulate ifonly a heterologous or extra endogenous DXS nucleic acid was present inthe cells.

In some embodiments, the production of isoprene by cells that contain aheterologous isoprene synthase nucleic acid is augmented by increasingthe amount of a MVA polypeptide expressed by the cells (FIGS. 19A and19B). Exemplary MVA pathways polypeptides include any of the followingpolypeptides: acetyl-CoA acetyltransferase (AA-CoA thiolase)polypeptides, 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase)polypeptides, 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoAreductase) polypeptides, mevalonate kinase (MVK) polypeptides,phosphomevalonate kinase (PMK) polypeptides, diphosphomevalonatedecarboxylase (MVD) polypeptides, phosphomevalonate decarboxylase (PMDC)polypeptides, isopentenyl phosphate kinase (IPK) polypeptides, IDIpolypeptides, and polypeptides (e.g., fusion polypeptides) having anactivity of two or more MVA pathway polypeptides. For example, one ormore MVA pathway nucleic acids can be introduced into the cells. In someembodiments, the cells contain the upper MVA pathway, which includesAA-CoA thiolase, HMG-CoA synthase, and HMG-CoA reductase nucleic acids.In some embodiments, the cells contain the lower MVA pathway, whichincludes MVK, PMK, MVD, and IDI nucleic acids. In some embodiments, thecells contain an entire MVA pathway that includes AA-CoA thiolase,HMG-CoA synthase, HMG-CoA reductase, MVK, PMK, MVD, and IDI nucleicacids. In some embodiments, the cells contain an entire MVA pathway thatincludes AA-CoA thiolase, HMG-CoA synthase, HMG-CoA reductase, MVK,PMDC, IPK, and IDI nucleic acids. The MVA pathway nucleic acids may beheterologous nucleic acids or duplicate copies of endogenous nucleicacids. In some embodiments, the amount of one or more MVA pathwaypolypeptides is increased by replacing the endogenous promoters orregulatory regions for the MVA pathway nucleic acids with otherpromoters and/or regulatory regions that result in greater transcriptionof the MVA pathway nucleic acids. In some embodiments, the cells containboth a heterologous nucleic acid encoding an isoprene synthasepolypeptide (e.g., a plant isoprene synthase nucleic acid) and aduplicate copy of an endogenous nucleic acid encoding an isoprenesynthase polypeptide.

For example, E. coli cells containing a nucleic acid encoding a kudzuisoprene synthase polypeptide and nucleic acids encoding Saccharomycescerevisiae MVK, PMK, MVD, and IDI polypeptides generated isoprene at arate of 6.67×10⁻⁴ mol/L_(broth)/OD₆₀₀/hr (see Example 8). Additionally,a 14 liter fermentation of E. coli cells with nucleic acids encodingEnterococcus faecalis AA-CoA thiolase, HMG-CoA synthase, and HMG-CoAreductase polypeptides produced 22 grams of mevalonic acid (anintermediate of the MVA pathway). A shake flask of these cells produced2-4 grams of mevalonic acid per liter. These results indicate thatheterologous MVA pathways nucleic acids are active in E. coli. E. colicells that contain nucleic acids for both the upper MVA pathway and thelower MVA pathway as well as a kudzu isoprene synthase (strain MCM 127)produced significantly more isoprene (874 ng/L) compared to E. colicells with nucleic acids for only the lower MVA pathway and the kudzuisoprene synthase (strain MCM 131) (see Table 3 and Example 8, partVIII).

In some embodiments, at least a portion of the cells maintain theheterologous isoprene synthase, DXS, IDI, and/or MVA pathway nucleicacid for at least about 5, 10, 20, 50, 75, 100, 200, 300, or more celldivisions in a continuous culture (such as a continuous culture withoutdilution). In some embodiments of any of the aspects described herein,the nucleic acid comprising the heterologous or duplicate copy of anendogenous isoprene synthase, DXS, IDI, and/or MVA pathway nucleic acidalso comprises a selective marker, such as a kanamycin, ampicillin,carbenicillin, gentamicin, hygromycin, phleomycin, bleomycin, neomycin,or chloramphenicol antibiotic resistance nucleic acid.

As indicated in Example 7, part VI, the amount of isoprene produced canbe further increased by adding yeast extract to the cell culture medium.In this example, the amount of isoprene produced was linearlyproportional to the amount of yeast extract in the cell medium for theconcentrations tested (FIG. 48C). Additionally, approximately 0.11 gramsof isoprene per liter of broth was produced from a cell medium withyeast extract and glucose (Example 7, part VIII). Both of theseexperiments used E. coli cells with kudzu isoprene synthase, S.cerevisia IDI, and E. coli DXS nucleic acids to produce isoprene.Increasing the amount of yeast extract in the presence of glucoseresulted in more isoprene being produced than increasing the amount ofglucose in the presence of yeast extract. Also, increasing the amount ofyeast extract allowed the cells to produce a high level of isoprene fora longer length of time and improved the health of the cells.

Isoprene production was also demonstrated using three types ofhydrolyzed biomass (bagasse, corn stover, and soft wood pulp) as thecarbon source (FIGS. 46A-C). E. coli cells with kudzu isoprene synthase,S. cerevisia IDI, and E. coli DXS nucleic acids produced as muchisoprene from these hydrolyzed biomass carbon sources as from theequivalent amount of glucose (e.g., 1% glucose, w/v). If desired, anyother biomass carbon source can be used in the compositions and methodsdescribed herein. Biomass carbon sources are desirable because they arecheaper than many conventional cell mediums, thereby facilitating theeconomical production of isoprene.

Additionally, invert sugar was shown to function as a carbon source forthe generation of isoprene (FIGS. 47C and 96-98). For example, 2.4 g/Lof isoprene was produced from cells expressing MVA pathway polypeptidesand a Kudzu isoprene synthase (Example 8, part XV). Glycerol was as alsoused as a carbon source for the generation of 2.2 mg/L of isoprene fromcells expressing a Kudzu isoprene synthase (Example 8, part XIV).Expressing a DXS nucleic acid, an IDI nucleic acid, and/or one or moreMVA pathway nucleic acids (such as nucleic acids encoding the entire MVApathway) in addition to an isoprene synthase nucleic acid may increasethe production of isoprene from glycerol.

In some embodiments, an oil is included in the cell medium. For example,B. subtilis cells containing a kudzu isoprene synthase nucleic acidproduced isoprene when cultured in a cell medium containing an oil and asource of glucose (Example 4, part III). As another example, E. colifadR atoC mutant cells containing the upper and lower MVA pathway pluskudzu isoprene synthase produced isoprene when cultured in a cell mediumcontaining palm oil and a source of glucose (Example 27, part II). Insome embodiments, more than one oil (such as 2, 3, 4, 5, or more oils)is included in the cell medium. While not intending to be bound to anyparticular theory, it is believed that (i) the oil may increase theamount of carbon in the cells that is available for conversion toisoprene, (ii) the oil may increase the amount of acetyl-CoA in thecells, thereby increasing the carbon flow through the MVA pathway,and/or (ii) the oil may provide extra nutrients to the cells, which isdesirable since much of the carbon in the cells is converted to isoprenerather than other products. In some embodiments, cells that are culturedin a cell medium containing oil naturally use the MVA pathway to produceisoprene or are genetically modified to contain nucleic acids for theentire MVA pathway. In some embodiments, the oil is partially orcompletely hydrolyzed before being added to the cell culture medium tofacilitate the use of the oil by the host cells.

One of the major hurdles to commercial production of small moleculessuch as isoprene in cells (e.g., bacteria) is the decoupling ofproduction of the molecule from growth of the cells. In some embodimentsfor the commercially viable production of isoprene, a significant amountof the carbon from the feedstock is converted to isoprene, rather thanto the growth and maintenance of the cells (“carbon efficiency”). Invarious embodiments, the cells convert greater than or about 0.0015,0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.12, 0.14, 0.16, 0.2, 0.3, 0.4,0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.5, 3.0, 3.5,4.0, 5.0, 6.0, 7.0, or 8.0% of the carbon in the cell culture mediuminto isoprene. In particular embodiments, a significant portion of thecarbon from the feedstock that is converted to downstream products isconverted to isoprene. As described further in Example 11, E. coli cellsexpressing MVA pathway and kudzu isoprene synthase nucleic acidsexhibited decoupling of the production of isoprene or the intermediatemevalonic acid from growth, resulting in high carbon efficiency. Inparticular, mevalonic acid was formed from cells expressing the upperMVA pathway from Enterococcus faecalis. Isoprene was formed from cellsexpressing the upper MVA pathway from Enterococcus faecalis, the lowerMVA pathway from Saccharomyces cerevisiae, and the isoprene synthasefrom Pueraria montana (Kudzu). This decoupling of isoprene or mevalonicacid production from growth was demonstrated in four different strainsof E. coli: BL21(LDE3), BL21(LDE3) Tuner, FM5, and MG1655. The first twoE. coli strains are B strains, and the latter two are K12 strains.Decoupling of production from growth was also demonstrated in a variantof MG1655 with ack and pta genes deleted. This variant also demonstratedless production of acetate.

Exemplary Polypeptides and Nucleic Acids

Various isoprene synthase, DXS, IDI, MVA pathway, hydrogenase,hydrogenase maturation or transcription factor polypeptides and nucleicacids can be used in the compositions and methods described herein.

In some embodiments, the fusion polypeptide includes part or all of afirst polypeptide (e.g., an isoprene synthase, DXS, IDI, MVA pathway,hydrogenase, hydrogenase maturation or transcription factor polypeptideor catalytically active fragment thereof) and may optionally includepart or all of a second polypeptide (e.g., a peptide that facilitatespurification or detection of the fusion polypeptide, such as a His-tag).In some embodiments, the fusion polypeptide has an activity of two ormore MVA pathway polypeptides (such as AA-CoA thiolase and HMG-CoAreductase polypeptides). In some embodiments, the polypeptide is anaturally-occurring polypeptide (such as the polypeptide encoded by anEnterococcus faecalis mvaE nucleic acid) that has an activity of two ormore MVA pathway polypeptides.

In various embodiments, a polypeptide has at least or about 50, 100,150, 175, 200, 250, 300, 350, 400, or more amino acids. In someembodiments, the polypeptide fragment contains at least or about 25, 50,75, 100, 150, 200, 300, or more contiguous amino acids from afull-length polypeptide and has at least or about 5%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of an activity of acorresponding full-length polypeptide. In particular embodiments, thepolypeptide includes a segment of or the entire amino acid sequence ofany naturally-occurring isoprene synthase, DXS, IDI, MVA pathway,hydrogenase, hydrogenase maturation or transcription factor polypeptide.In some embodiments, the polypeptide has one or more mutations comparedto the sequence of a wild-type (i.e., a sequence occurring in nature)isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenasematuration or transcription factor polypeptide.

In some embodiments, the polypeptide is an isolated polypeptide. In someembodiments, the polypeptide is a heterologous polypeptide.

In some embodiments, the nucleic acid is a recombinant nucleic acid. Insome embodiments, an isoprene synthase, DXS, IDI, MVA pathway,hydrogenase, hydrogenase maturation or transcription factor nucleic acidis operably linked to another nucleic acid encoding all or a portion ofanother polypeptide such that the recombinant nucleic acid encodes afusion polypeptide that includes an isoprene synthase, DXS, IDI, MVApathway, hydrogenase, hydrogenase maturation or transcription factorpolypeptide and all or part of another polypeptide (e.g., a peptide thatfacilitates purification or detection of the fusion polypeptide, such asa His-tag). In some embodiments, part or all of a recombinant nucleicacid is chemically synthesized. It is to be understood that mutations,including single nucleotide mutations, can occur within a nucleic acidas defined herein.

In some embodiments, the nucleic acid is a heterologous nucleic acid. Inparticular embodiments, the nucleic acid includes a segment of or theentire nucleic acid sequence of any naturally-occurring isoprenesynthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation ortranscription factor nucleic acid. In some embodiments, the nucleic acidincludes at least or about 50, 100, 150, 200, 300, 400, 500, 600, 700,800, or more contiguous nucleotides from a naturally-occurring isoprenesynthase nucleic acid DXS, IDI, MVA pathway, hydrogenase, hydrogenasematuration or transcription factor nucleic acid. In some embodiments,the nucleic acid has one or more mutations compared to the sequence of awild-type (i.e., a sequence occurring in nature) isoprene synthase, DXS,IDI, MVA pathway, hydrogenase, hydrogenase maturation or transcriptionfactor nucleic acid. In some embodiments, the nucleic acid has one ormore mutations (e.g., a silent mutation) that increase the transcriptionor translation of isoprene synthase, DXS, IDI, MVA pathway, hydrogenase,or transcription factor nucleic acid. In some embodiments, the nucleicacid is a degenerate variant of any nucleic acid encoding an isoprenesynthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation ortranscription factor polypeptide.

The accession numbers of exemplary isoprene synthase, DXS, IDI, and/orMVA pathway polypeptides and nucleic acids are listed in Appendix 1 (theaccession numbers of Appendix 1 and their corresponding sequences areherein incorporated by reference in their entireties, particularly withrespect to the amino acid and nucleic acid sequences of isoprenesynthase, DXS, IDI, and/or MVA pathway polypeptides and nucleic acids).The Kegg database also contains the amino acid and nucleic acidsequences of numerous exemplary isoprene synthase, DXS, IDI, and/or MVApathway polypeptides and nucleic acids (see, for example, the world-wideweb at “genome.jp/kegg/pathway/map/map00100.html” and the sequencestherein, which are each hereby incorporated by reference in theirentireties, particularly with respect to the amino acid and nucleic acidsequences of isoprene synthase, DXS, IDI, and/or MVA pathwaypolypeptides and nucleic acids). In some embodiments, one or more of theisoprene synthase, DXS, IDI, and/or MVA pathway polypeptides and/ornucleic acids have a sequence identical to a sequence publicly availableon Dec. 12, 2007 or Sep. 14, 2008 such as any of the sequences thatcorrespond to any of the accession numbers in Appendix 1 or any of thesequences present in the Kegg database. Additional exemplary isoprenesynthase, DXS, IDI, and/or MVA pathway polypeptides and nucleic acidsare described further below.

Exemplary Isoprene Synthase Polypeptides and Nucleic Acids

As noted above, isoprene synthase polypeptides convert dimethyl allyldiphosphate (DMAPP) into isoprene. Exemplary isoprene synthasepolypeptides include polypeptides, fragments of polypeptides, peptides,and fusions polypeptides that have at least one activity of an isoprenesynthase polypeptide. Standard methods can be used to determine whethera polypeptide has isoprene synthase polypeptide activity by measuringthe ability of the polypeptide to convert DMAPP into isoprene in vitro,in a cell extract, or in vivo. In an exemplary assay, cell extracts areprepared by growing a strain (e.g., the E. coli/pTrcKudzu straindescribed herein) in the shake flask method as described in Example 1.After induction is complete, approximately 10 mL of cells are pelletedby centrifugation at 7000×g for 10 minutes and re-suspended in 5 ml ofPEB without glycerol. The cells are lysed using a French Pressure cellusing standard procedures. Alternatively the cells are treated withlysozyme (Ready-Lyse lysozyme solution; EpiCentre) after a freeze/thawat −80° C.

Isoprene synthase polypeptide activity in the cell extract can bemeasured, for example, as described in Silver et al., J. Biol. Chem.270:13010-13016, 1995 and references therein, which are each herebyincorporated by reference in their entireties, particularly with respectto assays for isoprene synthase polypeptide activity. DMAPP (Sigma) isevaporated to dryness under a stream of nitrogen and re-hydrated to aconcentration of 100 mM in 100 mM potassium phosphate buffer pH 8.2 andstored at −20° C. To perform the assay, a solution of 5 μL of 1M MgCl₂,1 mM (250 μg/ml) DMAPP, 65 μL of Plant Extract Buffer (PEB) (50 mMTris-HCl, pH 8.0, 20 mM MgCl₂, 5% glycerol, and 2 mM DTT) is added to 25μL of cell extract in a 20 ml Headspace vial with a metal screw cap andteflon coated silicon septum (Agilent Technologies) and cultured at 37°C. for 15 minutes with shaking. The reaction is quenched by adding 200μL of 250 mM EDTA and quantified by GC/MS as described in Example 1,part II.

Exemplary isoprene synthase nucleic acids include nucleic acids thatencode a polypeptide, fragment of a polypeptide, peptide, or fusionpolypeptide that has at least one activity of an isoprene synthasepolypeptide. Exemplary isoprene synthase polypeptides and nucleic acidsinclude naturally-occurring polypeptides and nucleic acids from any ofthe source organisms described herein as well as mutant polypeptides andnucleic acids derived from any of the source organisms described herein.

In some embodiments, the isoprene synthase polypeptide or nucleic acidis from the family Fabaceae, such as the Faboideae subfamily. In someembodiments, the isoprene synthase polypeptide or nucleic acid is apolypeptide or nucleic acid from Pueraria montana (kudzu) (Sharkey etal., Plant Physiology 137: 700-712, 2005), Pueraria lobata, poplar (suchas Populus alba, Populus nigra, Populus trichocarpa, or Populusalba×tremula (CAC35696) Miller et al., Planta 213: 483-487, 2001) aspen(such as Populus tremuloides) Silver et al., JBC 270(22): 13010-1316,1995), or English Oak (Quercus robur) (Zimmer et al., WO 98/02550),which are each hereby incorporated by reference in their entireties,particularly with respect to isoprene synthase nucleic acids and theexpression of isoprene synthase polypeptides. Suitable isoprenesynthases include, but are not limited to, those identified by GenbankAccession Nos. AY341431, AY316691, AY279379, AJ457070, and AY182241,which are each hereby incorporated by reference in their entireties,particularly with respect to sequences of isoprene synthase nucleicacids and polypeptides. In some embodiments, the isoprene synthasepolypeptide or nucleic acid is not a naturally-occurring polypeptide ornucleic acid from Quercus robur (i.e., the isoprene synthase polypeptideor nucleic acid is an isoprene synthase polypeptide or nucleic acidother than a naturally-occurring polypeptide or nucleic acid fromQuercus robur). In some embodiments, the isoprene synthase nucleic acidor polypeptide is a naturally-occurring polypeptide or nucleic acid frompoplar. In some embodiments, the isoprene synthase nucleic acid orpolypeptide is not a naturally-occurring polypeptide or nucleic acidfrom poplar.

Exemplary DXS Polypeptides and Nucleic Acids

As noted above, 1-deoxy-D-xylulose-5-phosphate synthase (DXS)polypeptides convert pyruvate and D-glyceraldehyde-3-phosphate into1-deoxy-D-xylulose-5-phosphate. Exemplary DXS polypeptides includepolypeptides, fragments of polypeptides, peptides, and fusionspolypeptides that have at least one activity of a DXS polypeptide.Standard methods (such as those described herein) can be used todetermine whether a polypeptide has DXS polypeptide activity bymeasuring the ability of the polypeptide to convert pyruvate andD-glyceraldehyde-3-phosphate into 1-deoxy-D-xylulose-5-phosphate invitro, in a cell extract, or in vivo. Exemplary DXS nucleic acidsinclude nucleic acids that encode a polypeptide, fragment of apolypeptide, peptide, or fusion polypeptide that has at least oneactivity of a DXS polypeptide. Exemplary DXS polypeptides and nucleicacids include naturally-occurring polypeptides and nucleic acids fromany of the source organisms described herein as well as mutantpolypeptides and nucleic acids derived from any of the source organismsdescribed herein.

Exemplary IDI Polypeptides and Nucleic Acids

Isopentenyl diphosphate isomerase polypeptides (isopentenyl-diphosphatedelta-isomerase or IDI) catalyses the interconversion of isopentenyldiphosphate (IPP) and dimethyl allyl diphosphate (DMAPP) (e.g.,converting IPP into DMAPP and/or converting DMAPP into IPP). ExemplaryIDI polypeptides include polypeptides, fragments of polypeptides,peptides, and fusions polypeptides that have at least one activity of anIDI polypeptide. Standard methods (such as those described herein) canbe used to determine whether a polypeptide has IDI polypeptide activityby measuring the ability of the polypeptide to interconvert IPP andDMAPP in vitro, in a cell extract, or in vivo. Exemplary IDI nucleicacids include nucleic acids that encode a polypeptide, fragment of apolypeptide, peptide, or fusion polypeptide that has at least oneactivity of an IDI polypeptide. Exemplary IDI polypeptides and nucleicacids include naturally-occurring polypeptides and nucleic acids fromany of the source organisms described herein as well as mutantpolypeptides and nucleic acids derived from any of the source organismsdescribed herein.

Exemplary MVA Pathway Polypeptides and Nucleic Acids

Exemplary MVA pathway polypeptides include acetyl-CoA acetyltransferase(AA-CoA thiolase) polypeptides, 3-hydroxy-3-methylglutaryl-CoA synthase(HMG-CoA synthase) polypeptides, 3-hydroxy-3-methylglutaryl-CoAreductase (HMG-CoA reductase) polypeptides, mevalonate kinase (MVK)polypeptides, phosphomevalonate kinase (PMK) polypeptides,diphosphomevalonate decarboxylase (MVD) polypeptides, phosphomevalonatedecarboxylase (PMDC) polypeptides, isopentenyl phosphate kinase (IPK)polypeptides, IDI polypeptides, and polypeptides (e.g., fusionpolypeptides) having an activity of two or more MVA pathwaypolypeptides. In particular, MVA pathway polypeptides includepolypeptides, fragments of polypeptides, peptides, and fusionspolypeptides that have at least one activity of an MVA pathwaypolypeptide. Exemplary MVA pathway nucleic acids include nucleic acidsthat encode a polypeptide, fragment of a polypeptide, peptide, or fusionpolypeptide that has at least one activity of an MVA pathwaypolypeptide. Exemplary MVA pathway polypeptides and nucleic acidsinclude naturally-occurring polypeptides and nucleic acids from any ofthe source organisms described herein as well as mutant polypeptides andnucleic acids derived from any of the source organisms described herein.

In particular, acetyl-CoA acetyltransferase polypeptides (AA-CoAthiolase or AACT) convert two molecules of acetyl-CoA intoacetoacetyl-CoA. Standard methods (such as those described herein) canbe used to determine whether a polypeptide has AA-CoA thiolasepolypeptide activity by measuring the ability of the polypeptide toconvert two molecules of acetyl-CoA into acetoacetyl-CoA in vitro, in acell extract, or in vivo.

3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase or HMGS)polypeptides convert acetoacetyl-CoA into3-hydroxy-3-methylglutaryl-CoA. Standard methods (such as thosedescribed herein) can be used to determine whether a polypeptide hasHMG-CoA synthase polypeptide activity by measuring the ability of thepolypeptide to convert acetoacetyl-CoA into3-hydroxy-3-methylglutaryl-CoA in vitro, in a cell extract, or in vivo.

3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase or HMGR)polypeptides convert 3-hydroxy-3-methylglutaryl-CoA into mevalonate.Standard methods (such as those described herein) can be used todetermine whether a polypeptide has HMG-CoA reductase polypeptideactivity by measuring the ability of the polypeptide to convert3-hydroxy-3-methylglutaryl-CoA into mevalonate in vitro, in a cellextract, or in vivo.

Mevalonate kinase (MVK) polypeptides phosphorylates mevalonate to formmevalonate-5-phosphate. Standard methods (such as those describedherein) can be used to determine whether a polypeptide has MVKpolypeptide activity by measuring the ability of the polypeptide toconvert mevalonate into mevalonate-5-phosphate in vitro, in a cellextract, or in vivo.

Phosphomevalonate kinase (PMK) polypeptides phosphorylatesmevalonate-5-phosphate to form mevalonate-5-diphosphate. Standardmethods (such as those described herein) can be used to determinewhether a polypeptide has PMK polypeptide activity by measuring theability of the polypeptide to convert mevalonate-5-phosphate intomevalonate-5-diphosphate in vitro, in a cell extract, or in vivo.

Diphosphomevalonate decarboxylase (MVD or DPMDC) polypeptides convertmevalonate-5-diphosphate into isopentenyl diphosphate (IPP). Standardmethods (such as those described herein) can be used to determinewhether a polypeptide has MVD polypeptide activity by measuring theability of the polypeptide to convert mevalonate-5-diphosphate into IPPin vitro, in a cell extract, or in vivo.

Phosphomevalonate decarboxylase (PMDC) polypeptides convertmevalonate-5-phosphate into isopentenyl phosphate (IP). Standard methods(such as those described herein) can be used to determine whether apolypeptide has PMDC polypeptide activity by measuring the ability ofthe polypeptide to convert mevalonate-5-phosphate into IP in vitro, in acell extract, or in vivo.

Isopentenyl phosphate kinase (IPK) polypeptides phosphorylate isopentylphosphate (IP) to form isopentenyl diphosphate (IPP). Standard methods(such as those described herein) can be used to determine whether apolypeptide has IPK polypeptide activity by measuring the ability of thepolypeptide to convert IP into IPP in vitro, in a cell extract, or invivo.

Exemplary IDI polypeptides and nucleic acids are described above.

Exemplary Hydrogenase Polypeptides and Nucleic Acids

Hydrogenase polypeptides catalyze the reaction: 2H⁺+2e⁻

H₂. In vitro that reaction is reversible, but certain hydrogenases maywork in only one direction in vivo, either oxidizing H₂ or reducing H⁺.Hydrogenase polypeptides can be oxygen-sensitive, contain complex metalcofactors as part of their catalytic center and sometimes consist ofmultiple subunits, with hydrogenase gene expression sometimes involvingadditional accessory polypeptides, such as ‘maturation’ factors ortranscription regulatory factors (i.e., activators or repressors).Hydrogenases are classified into at least three broad groups based uponthe type of metal cofactor in their catalytic center: (1) nickel-iron(“NiFe”) hydrogenases have a nickel/iron cofactor; (2) iron-ironhydrogenases (“FeFe”) have an iron/iron cofactor; and (3)iron/sulfur-free (“Fe”) hydrogenases, which lack the 4Fe4S clustersfound in groups (1) and (2), have an iron cofactor and amethenyl-tetrahydromethanopterin electron carrier. See, e.g., Chung-JungChou et al., “Hydrogenesis in hyperthermophilic microorganisms:implications for biofuels,” Metabol. Eng. 10:394-404 (2008), and Gon111Vardar-Schara et al., “Metabolically engineered bacteria for producinghydrogen via fermentation,” Microbial Biotechnol. 1(2):107-125 (2008),both of which are incorporated herein by reference in their entireties,particularly with respect to the various types and classes ofhydrogenases. Although many organisms contain multiple hydrogenases, fewcontain genes for both NiFe and FeFe hydrogenases.

The catalytic center of NiFe hydrogenases consists of a nickel atom andan iron atom, each with two carbon monoxide (CO) and two cyanide (CN⁻)ligands. The NiFe hydrogenases all comprise at least a second subunitcontaining multiple iron-sulfur (Fe—S) centers for the transfer ofelectrons to and from the catalytic center. The NiFe hydrogenases can besubdivided into four main classes: (1) respiratory enzymes, which arepart of multi-enzyme systems that couple the oxidation of H₂ toreduction of terminal electron acceptors such as SO₄ ²⁻ or NO₃ ⁻ underanaerobic conditions, or to O₂ in aerobic microorganisms; (2) H₂sensors, which activate expression of the metabolically active NiFehydrogenases; (3) cytoplasmic hydrogenases, containing multiple subunitsable to utilize NADP⁺, which are readily reversible in vitro, but invivo may only oxidize H₂; and (4) membrane-bound, energy-conservingmulti-enzyme complexes also found in bacteria and Archaea. Chung-JungChou et al., “Hydrogenesis in hyperthermophilic microorganisms:implications for biofuels,” Metabol. Eng. 10:394-404 (2008).

The catalytic center of FeFe hydrogenases contains a catalytic “Hcluster” which coordinates a binuclear (FeFe) site bridged to a [4Fe-4S]center by a single protein (cysteine) ligand. The two iron atoms of thebinuclear center each have two carbon monoxide (CO) and two cyanide(CN⁻s) ligands, and are also bridged by two sulfur atoms which are partof a small organic molecule. Most FeFe hydrogenases are monomericenzymes of about 50 kilodaltons (kDa), and appear to function in vivoprimarily to dispose of excess reducing equivalents by reducing protonsto hydrogen gas. Chung-Jung Chou et al., “Hydrogenesis inhyperthermophilic microorganisms: implications for biofuels,” Metabol.Eng. 10:394-404 (2008).

The catalytic center of Fe hydrogenases was originally thought to havean active site based on an organic cofactor with no metals involved, butwas later shown to contain a mononuclear Fe atom. Despite thephylogenetic differences between the three types of hydrogenase, inaddition to at least one iron atom, all three groups of hydrogenasesalso contain at least one carbon monoxide (CO) ligand to the iron atomin their active sites, which facilitates the catalytic oxidation of H₂and the reduction of protons. Chung-Jung Chou et al., “Hydrogenesis inhyperthermophilic microorganisms: implications for biofuels,” Metabol.Eng. 10:394-404 (2008).

Exemplary hydrogenase polypeptides include, but are not limited to, theE. coli hydrogenase-1 (Hyd-1) polypeptides, E. coli hydrogenase-2(Hyd-2) polypeptides, E. coli hydrogenase-3 (Hyd-3) polypeptides, E.coli hydrogenase-4 (Hyd-4) polypeptides, E. coli formate hydrogen lyase(FHL) complex, which produces hydrogen gas from formate and CO₂ underanaerobic conditions at acidic pH (see, e.g., Akihito Yoshida et al.,“Efficient induction of formate hydrogen lyase of aerobically grownEscherichia coli in a three-step biohydrogen production process,” Appl.Microbiol. Biotechnol. 74:754-760 (2007), which is incorporated hereinby reference in its entirety, particularly with respect to the inductionof expression of formate hydrogen lyase in E. coli), Ralstonia eutrophaH16 hydrogenase (R. eutropha HoxH) Rhodococcus opacus MR11 hydrogenase(R. opacus HoxH) polypeptides, Synechosystis sp. PCC 6803 hydrogenase(Syn. PCC 6803 HoxH) polypeptides, Desulfovibrio gigas hydrogenase (D.gigas) polypeptides, and Desulfovibrio desulfuricans ATCC 7757hydrogenase (D. desulfuricans) polypeptides (see, e.g., GönülVardar-Schara et al., “Metabolically engineered bacteria for producinghydrogen via fermentation,” Microbial Biotechnol. 1(2):107-125 (2008),which is incorporated herein by reference in its entirety, particularlywith respect to the various types and classes of hydrogenases) andpolypeptides (e.g., fusion polypeptides) having an activity of two ormore hydrogenase polypeptides. In particular, hydrogenase polypeptidesinclude polypeptides, fragments of polypeptides, peptides, and fusionpolypeptides that have at least one activity of a hydrogenasepolypeptide. Exemplary hydrogenase nucleic acids include nucleic acidsthat encode a polypeptide, fragment of a polypeptide, peptide, or fusionpolypeptide that has at least one activity of a hydrogenase polypeptide,or at least one activity necessary for expression, processing, ormaturation of a hydrogenase polypeptide. Exemplary hydrogenasepolypeptides and nucleic acids include naturally-occurring polypeptidesand nucleic acids from any of the source organisms described herein aswell as mutant polypeptides and nucleic acids derived from any of thesource organisms described herein.

E. coli Hyd-3, which is part of the anaerobic formate hydrogen lyase(FHL) complex, is encoded by the hyc operon (comprising the hycA, hycB,hycC, hycD, hycE, hycF, hycG, hycH, and hycl genes). E. coli Hyd-4 isencoded by the hyf operon (comprising the hyfA, hyfB, hyfC, hyfD, hyfE,hyfF, hyfG, hyfH, hyfI, hyfJ, and hyfR genes). E. coli FHL is encoded bysix genes from the hyc operon (hycB, hycC, hycD, hycE, hycF and hycG)and the fdhF gene (encoding formate dehydrogenase H (Fdh-H)). Expressionof the FHL complex can further involve expression of pyruvate formatelyase (pfl), FhlA, a transcription factor that activates transcriptionof fdhF and the hyc operon, or deletion/inactivation of HycA, atranscription factor encoded by the hycA gene that negatively regulatestranscription of FHL. Co-production of isoprene and hydrogen can beimproved by expression or inactivation/deletion of additional proteinsinvolved in the regulation of gene expression for hydrogenases and otherenzymes, such as, for example, iron-sulfur complex transcriptionalregulator (iscR) (Kalim-Akhtar et al., “Deletion of iscR stimulatesrecombinant Clostridial Fe/Fe hydrogenase activity andH_(z)-accumulation in Escherichia coli BL21(DE3),” Appl. Microbiol.Biotechnol. 78:853-862 (2008), which is incorporated herein by referencein its entirety, particularly with reference to stimulation ofClostridial Fe/Fe hydrogenase activity and hydrogen accumulation in E.coli by deleting the iscR gene).

Exemplary ferredoxin-dependent hydrogenase polypeptides include, but arenot limited to, Clostridium acetobutulicum hydrogenase A (HydA) (see,e.g., P. W. King et al., “Functional studies of [FeFe] hydrogenasematuration in an Escherichia coli biosynthetic system,” J. Bacteriol.188(6):163-172 (2006), which is incorporated herein by reference in itsentirety, particularly with respect to production of hydrogen by HydAand three HydA-associated maturation enzymes (HydE, HydG, and HydF),which may be expressed alone or in conjunction with one or more of: (1)Bacillus subtilis NADPH ferredoxin oxidoreductase (NFOR) (see, e.g.,Viet et al., (2008)), which is incorporated herein by reference in itsentirety, particularly with respect to production of hydrogen by NFOR;see also PCT Publication No. WO/2007/089901, which is incorporatedherein by reference in its entirety, particularly with respect tooptimization of E. coli strains for production of hydrogen), Clostridiumkluyveri NADH ferredoxin oxidoreductase (RnfCDGEAB) (Henning Seedorf etal., “The genome of Clostridium kluyveri, a strict anaerobe with uniquemetabolic features,” Proc. Nat'l Acad. Sci. U.S.A. 105(6):2128-2133(2008), which is incorporated herein by reference in its entirety,particular with reference to NADH ferredoxin oxidoreductase, and withreference to components of the anaerobic ethanol-acetate fermentationpathway), or Clostridium pasteuranium ferredoxin oxidoreductase (Fdx);(2) glyceraldehyde-6-phosphate ferredoxin oxidoreductase (“GAPOR”); or(3) pyruvate ferredoxin oxidoreductase (“POR”), and polypeptides (e.g.,fusion polypeptides) having an activity of two or more hydrogenasepolypeptides or of one or more hydrogenase polypeptides and an activityof one or more ferredoxin-dependent oxidoreductases. In particular,ferredoxin-dependent hydrogenase polypeptides include polypeptides,fragments of polypeptides, peptides, and fusion polypeptides that haveat least one activity of a ferredoxin-dependent hydrogenase polypeptide.

Exemplary NADPH-dependent hydrogenase polypeptides include, but are notlimited to thermophilic hydrogenase polypeptides such as Pyrococcusfuriosus hydrogenase (see, e.g., J. Woodward et al., “Enzymaticproduction of biohydrogen,” Nature 405(6790):1014-1015 (2000)), andpolypeptides (e.g., fusion polypeptides) having an activity of two ormore NADPH-dependent hydrogenase polypeptides. In particular,NADPH-dependent hydrogenase polypeptides include polypeptides, fragmentsof polypeptides, peptides, and fusion polypeptides that have at leastone activity of a NADPH-dependent hydrogenase polypeptide.

Exemplary oxygen-tolerant or oxygen-insensitive hydrogenases include,but are not limited to, Rubrivivax gelatinosus hydrogenase (see, e.g.,P. C. Maness et al., “Characterization of the oxygen tolerance of ahydrogenase linked to a carbon monoxide oxidation pathway in Rubrivivaxgelatinosus,” Appl. Environ. Microbiol. 68(6):2633-2636 (2002), which isincorporated herein by reference in its entirety, particularly withrespect to R. gelatinosus hydrogenase), and Ralstonia eutrophahydrogenase polypeptides (see, e.g., T. Burgdorf et al.,“[NiFe]-hydrogenases of Ralstonia eutropha H16: modular enzymes foroxygen-tolerant biological hydrogen oxidation,” J. Mol. Microbiol.Biotechnol. 10(2-4):181-196 (2005), which is incorporated herein byreference in its entirety, particularly with respect to R. eutrophahydrogenase polypeptides). Alternatively, heterologous nucleic acidsencoding hydrogenase polypeptides can be mutagenized and screened forO₂-tolerance or O₂-insensitivity using standard methods and assays (see,e.g., L. E. Nagy et al., “Application of gene-shuffling for the rapidgeneration of novel [FeFe]-hydrogenase libraries,” Biotechnol. Letts.29(3)421-430 (2007), which is incorporated herein by reference,particularly with respect to mutagenesis and screening for oxygentolerant hydrogenase polypeptides).

Standard methods (such as those described herein) can be used todetermine whether a polypeptide has hydrogenase activity by measuringthe ability of the polypeptide to produce hydrogen gas in vitro, in acell extract, or in vivo.

Exemplary Polypeptides and Nucleic Acids for Genes Related to Productionof Fermentation Side Products

In addition to expressing or over-expressing heterologous or nativehydrogenases in E. coli, co-production of isoprene and hydrogen can beimproved by inactivation of anaerobic biosynthetic pathways, therebyblocking the carbon flow to a variety of metabolites (i.e., fermentationside products) produced under oxygen-limited or anaerobic conditions,including, but not limited to, lactate, acetate, pyruvate, ethanol,succinate, and glycerol. Exemplary polypeptides involved in theproduction of fermentation side products include formate dehydrogenaseN, alpha subunit (fdnG), formate dehydrogenase O, large subunit (fdoG),nitrate reductase (narG), formate transporter A (focA), formatetransporter B (focB), pyruvate oxidase (poxB), pyruvate dehydrogenase E1component ackA/pta (aceE), alcohol dehydrogenase (adhE), fumaratereductase membrane protein (frdC), and lactate dehydrogenase (ldhA).See, e.g., Toshinori Maeda et al., “Enhanced hydrogen production fromglucose by metabolically engineered Escherichia coli,” Appl. Microbiol.Biotechnol. 77(4):879-890 (2007), which is incorporated by reference inits entirety, particularly with respect to production of E. coli strainswith modified glucose metabolism. Exemplary polypeptides involved in theregulation or expression of genes involved in the production offermentation side products that may also be inactivated to improveco-production of isoprene and hydrogen include, but are not limited to,repressor of formate hydrogen lyase (hycA), fumarate reductase regulator(fnr), acetyl-coenzyme A synthetase (acs), and formate dehydrogenaseregulatory protein (hycA), which regulates expression of thetranscriptional regulator fhlA (formate hydrogen lyase transcriptionalactivator).

Exemplary Polypeptides and Nucleic Acids for Genes Related to HydrogenRe-Uptake

Exemplary polypeptides involved in hydrogen re-uptake that may also beinactivated to improve co-production of isoprene and hydrogen include,but are not limited to, E. coli hydrogenase-1 (Hyd-1) (hya operon) andE. coli hydrogenase-2 (Hyd-2) (hyb operon). E. coli Hyd-1 is encoded bythe hya operon (comprising the hyaA, hyaB, hyaC, hyaD, hyaE, and hyaFgenes). E. coli Hyd-2 is encoded by the hyb operon (comprising the hybA,hybB, hybC, hybD, hybE, hybF, hybG, and hybO genes).

Exemplary Methods for Isolating Nucleic Acids

Isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenasematuration and/or transcription factor nucleic acids can be isolatedusing standard methods. Methods of obtaining desired nucleic acids froma source organism of interest (such as a bacterial genome) are commonand well known in the art of molecular biology (see, for example, WO2004/033646 and references cited therein, which are each herebyincorporated by reference in their entireties, particularly with respectto the isolation of nucleic acids of interest). For example, if thesequence of the nucleic acid is known (such as any of the known nucleicacids described herein), suitable genomic libraries may be created byrestriction endonuclease digestion and may be screened with probescomplementary to the desired nucleic acid sequence. Once the sequence isisolated, the DNA may be amplified using standard primer directedamplification methods such as polymerase chain reaction (PCR) (U.S. Pat.No. 4,683,202, which is incorporated by reference in its entirety,particularly with respect to PCR methods) to obtain amounts of DNAsuitable for transformation using appropriate vectors.

Alternatively, isoprene synthase, DXS, IDI, MVA pathway, hydrogenase,hydrogenase maturation and/or transcription factor nucleic acids (suchas any isoprene synthase, DXS, IDI, MVA pathway, hydrogenase,hydrogenase maturation and/or transcription factor nucleic acids with aknown nucleic acid sequence) can be chemically synthesized usingstandard methods.

Additional isoprene synthase, DXS, IDI, MVA pathway, hydrogenase,hydrogenase maturation and/or transcription factor polypeptides andnucleic acids which may be suitable for use in the compositions andmethods described herein can be identified using standard methods. Forexample, cosmid libraries of the chromosomal DNA of organisms known toproduce isoprene naturally can be constructed in organisms such as E.coli, and then screened for isoprene production. In particular, cosmidlibraries may be created where large segments of genomic DNA (35-45 kb)are packaged into vectors and used to transform appropriate hosts.Cosmid vectors are unique in being able to accommodate large quantitiesof DNA. Generally cosmid vectors have at least one copy of the cos DNAsequence which is needed for packaging and subsequent circularization ofthe heterologous DNA. In addition to the cos sequence, these vectorsalso contain an origin of replication such as ColEI and drug resistancemarkers such as a nucleic acid resistant to ampicillin or neomycin.Methods of using cosmid vectors for the transformation of suitablebacterial hosts are well described in Sambrook et al., MolecularCloning: A Laboratory Manual, 2^(nd) ed., Cold Spring Harbor, 1989,which is hereby incorporated by reference in its entirety, particularlywith respect to transformation methods.

Typically to clone cosmids, heterologous DNA is isolated using theappropriate restriction endonucleases and ligated adjacent to the cosregion of the cosmid vector using the appropriate ligases. Cosmidvectors containing the linearized heterologous DNA are then reacted witha DNA packaging vehicle such as bacteriophage. During the packagingprocess, the cos sites are cleaved and the heterologous DNA is packagedinto the head portion of the bacterial viral particle. These particlesare then used to transfect suitable host cells such as E. coli. Onceinjected into the cell, the heterologous DNA circularizes under theinfluence of the cos sticky ends. In this manner, large segments ofheterologous DNA can be introduced and expressed in host cells.

Additional methods for obtaining isoprene synthase, DXS, IDI, MVApathway, hydrogenase, hydrogenase maturation and/or transcription factornucleic acids include screening a metagenomic library by assay (such asthe headspace assay described herein) or by PCR using primers directedagainst nucleotides encoding for a length of conserved amino acids (forexample, at least 3 conserved amino acids). Conserved amino acids can beidentified by aligning amino acid sequences of known isoprene synthase,DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/ortranscription factor polypeptides. Conserved amino acids for isoprenesynthase polypeptides can be identified based on aligned sequences ofknown isoprene synthase polypeptides. An organism found to produceisoprene naturally can be subjected to standard protein purificationmethods (which are well known in the art) and the resulting purifiedpolypeptide can be sequenced using standard methods. Other methods arefound in the literature (see, for example, Julsing et al., Applied.Microbiol. Biotechnol. 75: 1377-84, 2007; Withers et al., Appl EnvironMicrobiol. 73(19):6277-83, 2007, which are each hereby incorporated byreference in their entireties, particularly with respect toidentification of nucleic acids involved in the synthesis of isoprene).

Additionally, standard sequence alignment and/or structure predictionprograms can be used to identify additional DXS, IDI, MVA pathway,hydrogenase, hydrogenase maturation and/or transcription factorpolypeptides and nucleic acids based on the similarity of their primaryand/or predicted polypeptide secondary structure with that of known DXS,IDI, MVA pathway, hydrogenase, hydrogenase maturation and/ortranscription factor polypeptides and nucleic acids. Standard databasessuch as the swissprot-trembl database (world-wide web at “expasy.org”,Swiss Institute of Bioinformatics Swiss-Prot group CMU-1 rue MichelServet CH-1211 Geneva 4, Switzerland) can also be used to identifyisoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenasematuration and/or transcription regulatory polypeptides and nucleicacids. The secondary and/or tertiary structure of an isoprene synthase,DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/ortranscription factor polypeptide can be predicted using the defaultsettings of standard structure prediction programs, such asPredictProtein (630 West, 168 Street, BB217, New York, N.Y. 10032, USA).Alternatively, the actual secondary and/or tertiary structure of anisoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenasematuration and/or transcription factor polypeptide can be determinedusing standard methods. Additional isoprene synthase, DXS, IDI, MVApathway, hydrogenase, hydrogenase maturation and/or transcription factornucleic acids can also be identified by hybridization to probesgenerated from known isoprene synthase, DXS, IDI, MVA pathway,hydrogenase, hydrogenase maturation and/or transcription factor nucleicacids.

Exemplary Promoters and Vectors

Any of the isoprene synthase, DXS, IDI, MVA pathway, hydrogenase,hydrogenase maturation and/or transcription factor nucleic acidsdescribed herein can be included in one or more vectors. Accordingly,also described herein are vectors with one more nucleic acids encodingany of the isoprene synthase, DXS, IDI, MVA pathway, hydrogenase,hydrogenase maturation and/or transcription factor polypeptides that aredescribed herein. In some embodiments, the vector contains a nucleicacid under the control of an expression control sequence.

In some embodiments, the vector contains a selective marker orselectable marker. Markers useful in vector systems for transformationof Trichoderma are known in the art (see, e.g., Finkelstein, Chapter 6in Biotechnology of Filamentous Fungi, Finkelstein et al., Eds.Butterworth-Heinemann, Boston, Mass., Chap. 6., 1992; and Kinghorn etal., Applied Molecular Genetics of Filamentous Fungi, Blackie Academicand Professional, Chapman and Hall, London, 1992, which are each herebyincorporated by reference in their entireties, particularly with respectto selective markers). In some embodiments, the selective marker is theamdS nucleic acid, which encodes the enzyme acetamidase, allowingtransformed cells to grow on acetamide as a nitrogen source. The use ofan A. nidulans amdS nucleic acid as a selective marker is described inKelley et al., EMBO J. 4:475-479, 1985 and Penttila et al., Gene61:155-164, 1987 (which are each hereby incorporated by reference intheir entireties, particularly with respect to selective markers). Insome embodiments, an isoprene synthase, DXS, IDI, MVA pathway,hydrogenase, hydrogenase maturation, or transcription regulatory nucleicacid integrates into a chromosome of the cells without a selectivemarker.

Suitable vectors are those which are compatible with the host cellemployed. Suitable vectors can be derived, for example, from abacterium, a virus (such as bacteriophage T7 or a M-13 derived phage), acosmid, a yeast, or a plant. Protocols for obtaining and using suchvectors are known to those in the art (see, for example, Sambrook etal., Molecular Cloning: A Laboratory Manual, 2^(nd) ed., Cold SpringHarbor, 1989, which is hereby incorporated by reference in its entirety,particularly with respect to the use of vectors).

Promoters are well known in the art. Any promoter that functions in thehost cell can be used for expression of an isoprene synthase, DXS, IDI,MVA pathway, hydrogenase, hydrogenase maturation and/or transcriptionfactor nucleic acid in the host cell. Initiation control regions orpromoters, which are useful to drive expression of isoprene synthase,DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/ortranscription factor nucleic acids in various host cells are numerousand familiar to those skilled in the art (see, for example, WO2004/033646 and references cited therein, which are each herebyincorporated by reference in their entireties, particularly with respectto vectors for the expression of nucleic acids of interest). Virtuallyany promoter capable of driving these nucleic acids can be usedincluding, but not limited to, CYCl, HIS3, GALL, GAL10, ADH1, PGK, PHO5,GAPDH, ADCI, TRP1, URA3, LEU2, ENO, and TPI (useful for expression inSaccharomyces); AOX1 (useful for expression in Pichia); and lac, trp,λP_(L), λP_(R), T7, tac, and trc (useful for expression in E. coli).

In some embodiments, a glucose isomerase promoter is used (see, forexample, U.S. Pat. No. 7,132,527 and references cited therein, which areeach hereby incorporated by reference in their entireties, particularlywith respect promoters and plasmid systems for expressing polypeptidesof interest). Reported glucose isomerase promoter mutants can be used tovary the level of expression of the polypeptide encoded by a nucleicacid operably linked to the glucose isomerase promoter (U.S. Pat. No.7,132,527). In various embodiments, the glucose isomerase promoter iscontained in a low, medium, or high copy plasmid (U.S. Pat. No.7,132,527).

In various embodiments, an isoprene synthase, DXS, IDI, MVA pathway,hydrogenase, hydrogenase maturation and/or transcription factor nucleicacid is contained in a low copy plasmid (e.g., a plasmid that ismaintained at about 1 to about 4 copies per cell), medium copy plasmid(e.g., a plasmid that is maintained at about 10 to about 15 copies percell), or high copy plasmid (e.g., a plasmid that is maintained at about50 or more copies per cell). In some embodiments, the heterologous orextra endogenous isoprene synthase, DXS, IDI, MVA pathway, hydrogenase,hydrogenase maturation and/or transcription factor nucleic acid isoperably linked to a T7 promoter. In some embodiments, the heterologousor extra endogenous isoprene synthase, DXS, IDI, MVA pathway,hydrogenase, hydrogenase maturation and/or transcription factor nucleicacid operably linked to a T7 promoter is contained in a medium or highcopy plasmid. In some embodiments, the heterologous or extra endogenousisoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenasematuration and/or transcription factor nucleic acid is operably linkedto a Trc promoter. In some embodiments, the heterologous or extraendogenous isoprene synthase, DXS, IDI, MVA pathway, hydrogenase,hydrogenase maturation and/or transcription factor nucleic acid operablylinked to a Trc promoter is contained in a medium or high copy plasmid.In some embodiments, the heterologous or extra endogenous isoprenesynthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturationand/or transcription factor nucleic acid is operably linked to a Lacpromoter. In some embodiments, the heterologous or extra endogenousisoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenasematuration and/or transcription factor nucleic acid operably linked to aLac promoter is contained in a low copy plasmid. In some embodiments,the heterologous or extra endogenous isoprene synthase, DXS, IDI, MVApathway, hydrogenase, hydrogenase maturation and/or transcription factornucleic acid is operably linked to an endogenous promoter, such as anendogenous Escherichia, Panteoa, Bacillus, Yarrowia, Streptomyces, orTrichoderma promoter or an endogenous alkaline serine protease, isoprenesynthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturationand/or transcription factor promoter. In some embodiments, theheterologous or extra endogenous isoprene synthase, DXS, IDI, MVApathway, hydrogenase, hydrogenase maturation and/or transcription factornucleic acid operably linked to an endogenous promoter is contained in ahigh copy plasmid. In some embodiments, the vector is a replicatingplasmid that does not integrate into a chromosome in the cells. In someembodiments, part or all of the vector integrates into a chromosome inthe cells.

In some embodiments, the vector is any vector which when introduced intoa fungal host cell is integrated into the host cell genome and isreplicated. Reference is made to the Fungal Genetics Stock CenterCatalogue of Strains (FGSC, the world-wide web at “fgsc.net” and thereferences cited therein, which are each hereby incorporated byreference in their entireties, particularly with respect to vectors) fora list of vectors. Additional examples of suitable expression and/orintegration vectors are provided in Sambrook et al., Molecular Cloning:A Laboratory Manual, 2^(nd) ed., Cold Spring Harbor, 1989, CurrentProtocols in Molecular Biology (F. M. Ausubel et al. (eds) 1987,Supplement 30, section 7.7.18); van den Hondel et al. in Bennett andLasure (Eds.) More Gene Manipulations in Fungi, Academic Press pp.396-428, 1991; and U.S. Pat. No. 5,874,276, which are each herebyincorporated by reference in their entireties, particularly with respectto vectors. Particularly useful vectors include pFB6, pBR322, PUC18,pUC100, and pENTR/D.

In some embodiments, an isoprene synthase, DXS, IDI, MVA pathway,hydrogenase, hydrogenase maturation and/or transcription factor nucleicacid is operably linked to a suitable promoter that showstranscriptional activity in a fungal host cell. The promoter may bederived from one or more nucleic acids encoding a polypeptide that iseither endogenous or heterologous to the host cell. In some embodiments,the promoter is useful in a Trichoderma host. Suitable non-limitingexamples of promoters include cbh1, cbh2, egl1, egl2, pepA, hibl, hfb2,xynl, and amy. In some embodiments, the promoter is one that is nativeto the host cell. For example, in some embodiments when T. reesei is thehost, the promoter is a native T. reesei promoter. In some embodiments,the promoter is T. reesei cbh1, which is an inducible promoter and hasbeen deposited in GenBank under Accession No. D86235, which isincorporated by reference in its entirety, particularly with respect topromoters. In some embodiments, the promoter is one that is heterologousto the fungal host cell. Other examples of useful promoters includepromoters from the genes of A. awamori and A. niger glucoamylase (glaA)(Nunberg et al., Mol. Cell. Biol. 4:2306-2315, 1984 and Boel et al.,EMBO J. 3:1581-1585, 1984, which are each hereby incorporated byreference in their entireties, particularly with respect to promoters);Aspergillus niger alpha amylases, Aspergillus oryzae TAKA amylase, T.reesei xlnl, and the T. reesei cellobiohydrolase 1 (EP 137280, which isincorporated by reference in its entirety, particularly with respect topromoters).

In some embodiments, the expression vector also includes a terminationsequence. Termination control regions may also be derived from variousgenes native to the host cell. In some embodiments, the terminationsequence and the promoter sequence are derived from the same source. Inanother embodiment, the termination sequence is endogenous to the hostcell. A particularly suitable terminator sequence is cbh1 derived from aTrichoderma strain (such as T. reesei). Other useful fungal terminatorsinclude the terminator from an A. niger or A. awamori glucoamylasenucleic acid (Nunberg et al., Mol. Cell Biol. 4:2306-2315, 1984 and Boelet al., EMBO J. 3:1581-1585, 1984; which are each hereby incorporated byreference in their entireties, particularly with respect to fungalterminators). Optionally, a termination site may be included. Foreffective expression of the polypeptides, DNA encoding the polypeptideare linked operably through initiation codons to selected expressioncontrol regions such that expression results in the formation of theappropriate messenger RNA.

In some embodiments, the promoter, coding, region, and terminator alloriginate from the isoprene synthase, DXS, IDI, MVA pathway,hydrogenase, hydrogenase maturation and/or transcription factor nucleicacid to be expressed. In some embodiments, the coding region for anisoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenasematuration and/or transcription factor nucleic acid is inserted into ageneral-purpose expression vector such that it is under thetranscriptional control of the expression construct promoter andterminator sequences. In some embodiments, genes or part thereof areinserted downstream of the strong cbh1 promoter.

An isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenasematuration and/or transcription factor nucleic acid can be incorporatedinto a vector, such as an expression vector, using standard techniques(Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor, 1982, which is hereby incorporated by reference in its entirety,particularly with respect to the screening of appropriate DNA sequencesand the construction of vectors). Methods used to ligate the DNAconstruct comprising a nucleic acid of interest (such as an isoprenesynthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturationand/or transcription factor nucleic acid), a promoter, a terminator, andother sequences and to insert them into a suitable vector are well knownin the art. For example, restriction enzymes can be used to cleave theisoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenasematuration and/or transcription factor nucleic acid and the vector.Then, the compatible ends of the cleaved isoprene synthase, DXS, IDI,MVA pathway, hydrogenase, hydrogenase maturation and/or transcriptionfactor nucleic acid and the cleaved vector can be ligated Linking isgenerally accomplished by ligation at convenient restriction sites. Ifsuch sites do not exist, the synthetic oligonucleotide linkers are usedin accordance with conventional practice (see, Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2^(nd) ed., Cold Spring Harbor,1989, and Bennett and Lasure, More Gene Manipulations in Fungi, AcademicPress, San Diego, pp 70-76, 1991, which are each hereby incorporated byreference in their entireties, particularly with respect tooligonucleotide linkers). Additionally, vectors can be constructed usingknown recombination techniques (e.g., Invitrogen Life Technologies,Gateway Technology).

In some embodiments, it may be desirable to over-express isoprenesynthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturationand/or transcription factor nucleic acids at levels far higher thancurrently found in naturally-occurring cells. This result may beaccomplished by the selective cloning of the nucleic acids encodingthose polypeptides into multicopy plasmids or placing those nucleicacids under a strong inducible or constitutive promoter. Methods forover-expressing desired polypeptides are common and well known in theart of molecular biology and examples may be found in Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2^(nd) ed., Cold Spring Harbor,1989, which is hereby incorporated by reference in its entirety,particularly with respect to cloning techniques.

In some embodiments, it may be desirable to under-express (e.g., mutate,inactivate, or delete) isoprene synthase, DXS, IDI, MVA pathway,hydrogenase, hydrogenase maturation, or transcription factorpolypeptide-encoding nucleic acids at levels far below that thosecurrently found in naturally-occurring cells. This result may beaccomplished by the mutation or inactivation of transcriptionalregulatory proteins required for expression of isoprene synthase, DXS,IDI, MVA pathway, hydrogenase, hydrogenase maturation and/ortranscription factor nucleic acids, by deletion of the isoprenesynthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturationand/or transcription factor nucleic acids, or by placing those nucleicacids under the control of a strong repressible promoter. Methods formutating, inactivating, or deleting desired polypeptides are common andwell known in the art of molecular biology and examples may be found inSambrook et al., Molecular Cloning: A Laboratory Manual, 2^(nd) ed.,Cold Spring Harbor, 1989, which is hereby incorporated by reference inits entirety, particularly with respect to cloning and mutagenesistechniques.

The following resources include descriptions of additional generalmethodology useful in accordance with the compositions and methodsdescribed herein: Kreigler, Gene Transfer and Expression; A LaboratoryManual, 1990 and Ausubel et al., Eds. Current Protocols in MolecularBiology, 1994, which are each hereby incorporated by reference in theirentireties, particularly with respect to molecular biology and cloningtechniques.

Exemplary Source Organisms

Isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenasematuration and/or transcription factor nucleic acids (and their encodedpolypeptides) can be obtained from any organism that naturally containsisoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenasematuration and/or transcription factor nucleic acids. As noted above,isoprene is formed naturally by a variety of organisms, such asbacteria, yeast, plants, and animals. Organisms contain the MVA pathway,DXP pathway, or both the MVA and DXP pathways for producing isoprene(FIGS. 19A and 19B). Thus, DXS nucleic acids can be obtained, e.g., fromany organism that contains the DXP pathway or contains both the MVA andDXP pathways. IDI and isoprene synthase nucleic acids can be obtained,e.g., from any organism that contains the MVA pathway, DXP pathway, orboth the MVA and DXP pathways. MVA pathway nucleic acids can beobtained, e.g., from any organism that contains the MVA pathway orcontains both the MVA and DXP pathways. Hydrogenase nucleic acids can beobtained, e.g., from any organism that oxidizes hydrogen or reduceshydrogen ions. Fermentation side product genes can be obtained oridentified, e.g., from any organism that undergoes oxygen-limited oranaerobic respiration, such as glycolysis.

In some embodiments, the nucleic acid sequence of the isoprene synthase,DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/ortranscription factor nucleic is identical to the sequence of a nucleicacid that is produced by any of the following organisms in nature. Insome embodiments, the amino acid sequence of the isoprene synthase, DXS,IDI, MVA pathway, hydrogenase, hydrogenase maturation and/ortranscription factor polypeptide is identical to the sequence of apolypeptide that is produced by any of the following organisms innature. In some embodiments, the isoprene synthase, DXS, IDI, MVApathway, hydrogenase, hydrogenase maturation and/or transcription factornucleic acid or polypeptide is a mutant nucleic acid or polypeptidederived from any of the organisms described herein. As used herein,“derived from” refers to the source of the nucleic acid or polypeptideinto which one or more mutations is introduced. For example, apolypeptide that is “derived from a plant polypeptide” refers topolypeptide of interest that results from introducing one or moremutations into the sequence of a wild-type (i.e., a sequence occurringin nature) plant polypeptide.

In some embodiments, the source organism is a fungus, examples of whichare species of Aspergillus such as A. oryzae and A. niger, species ofSaccharomyces such as S. cerevisiae, species of Schizosaccharomyces suchas S. pombe, and species of Trichoderma such as T. reesei. In someembodiments, the source organism is a filamentous fungal cell. The term“filamentous fungi” refers to all filamentous forms of the subdivisionEumycotina (see, Alexopoulos, C. J. (1962), Introductory Mycology,Wiley, New York). These fungi are characterized by a vegetative myceliumwith a cell wall composed of chitin, cellulose, and other complexpolysaccharides. The filamentous fungi are morphologically,physiologically, and genetically distinct from yeasts. Vegetative growthby filamentous fungi is by hyphal elongation and carbon catabolism isobligatory aerobic. The filamentous fungal parent cell may be a cell ofa species of, but not limited to, Trichoderma, (e.g., Trichodermareesei, the asexual morph of Hypocrea jecorina, previously classified asT. longibrachiatum, Trichoderma viride, Trichoderma koningii,Trichoderma harzianum) (Sheir-Neirs et al., Appl. Microbiol. Biotechnol20: 46-53, 1984; ATCC No. 56765 and ATCC No. 26921); Penicillium sp.,Humicola sp. (e.g., H. insolens, H. lanuginose, or H. grisea);Chrysosporium sp. (e.g., C. lucknowense), Gliocladium sp., Aspergillussp. (e.g., A. oryzae, A. niger, A. sojae, A. japonicus, A. nidulans, orA. awamori) (Ward et al., Appl. Microbiol. Biotechnol. 39: 7380743, 1993and Goedegebuur et al., Genet. 41: 89-98, 2002), Fusarium sp., (e.g., F.roseum, F. graminum F. cerealis, F. oxysporuim, or F. venenatum),Neurospora sp., (e.g., N. crassa), Hypocrea sp., Mucor sp., (e.g., Mmiehei), Rhizopus sp. and Emericella sp. (see also, Innis et al., Sci.228: 21-26, 1985). The term “Trichoderma” or “Trichoderma sp.” or“Trichoderma spp.” refer to any fungal genus previously or currentlyclassified as Trichoderma.

In some embodiments, the fungus is A. nidulans, A. awamori, A. oryzae,A. aculeatus, A. niger, A. japonicus, T. reesei, T. viride, F.oxysporum, or F. solani. Aspergillus strains are disclosed in Ward etal., Appl. Microbiol. Biotechnol. 39:738-743, 1993 and Goedegebuur etal., Curr Gene 41:89-98, 2002, which are each hereby incorporated byreference in their entireties, particularly with respect to fungi. Inparticular embodiments, the fungus is a strain of Trichoderma, such as astrain of T. reesei. Strains of T. reesei are known and non-limitingexamples include ATCC No. 13631, ATCC No. 26921, ATCC No. 56764, ATCCNo. 56765, ATCC No. 56767, and NRRL 15709, which are each herebyincorporated by reference in their entireties, particularly with respectto strains of T. reesei. In some embodiments, the host strain is aderivative of RL-P37. RL-P37 is disclosed in Sheir-Neiss et al., Appl.Microbiol. Biotechnology 20:46-53, 1984, which is hereby incorporated byreference in its entirety, particularly with respect to strains of T.reesei.

In some embodiments, the source organism is a yeast, such asSaccharomyces sp., Schizosaccharomyces sp., Pichia sp., or Candida sp.In some embodiments, the Saccharomyces sp. is Saccharomyces cerevisiae.

In some embodiments, the source organism is a bacterium, such as strainsof Bacillus such as B. lichenformis or B. subtilis, strains of Pantoeasuch as P. citrea, strains of Pseudomonas such as P. alcaligenes, P.putida, or P. fluorescens, strains of Streptomyces such as S. lividansor S. rubiginosus, strains of Corynebacterium sp. such asCorynebacterium glutamicum, strains of Rhodopseudomonas sp. such asRhodopseudomonas palustris, or strains of Escherichia such as E. coli.

As used herein, “the genus Bacillus” includes all species within thegenus “Bacillus,” as known to those of skill in the art, including butnot limited to B. subtilis, B. licheniformis, B. lentus, B. brevis, B.stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii,B. halodurans, B. megaterium, B. coagulans, B. circulans, B. lautus, andB. thuringiensis. It is recognized that the genus Bacillus continues toundergo taxonomical reorganization. Thus, it is intended that the genusinclude species that have been reclassified, including but not limitedto such organisms as B. stearothermophilus, which is now named“Geobacillus stearothermophilus.” The production of resistant endosporesin the presence of oxygen is considered the defining feature of thegenus Bacillus, although this characteristic also applies to therecently named Alicyclobacillus, Amphibacillus, Aneurinibacillus,Anoxybacillus, Brevibacillus, Filobacillus, Gracilibacillus,Halobacillus, Paenibacillus, Salibacillus, Thermobacillus, Ureibacillus,and Virgibacillus.

In some embodiments, the source organism is a gram-positive bacterium.Non-limiting examples include strains of Streptomyces (e.g., S.lividans, S. coelicolor, or S. griseus) and Bacillus. In someembodiments, the source organism is a gram-negative bacterium, such asE. coli., Rhodopseudomonas sp. such as Rhodopseudomonas palustris, orPseudomonas sp., such as P. alcaligenes, P. putida, or P. fluorescens.

In some embodiments, the source organism is a plant, such as a plantfrom the family Fabaceae, such as the Faboideae subfamily. In someembodiments, the source organism is kudzu, poplar (such as Populusalba×tremula CAC35696), aspen (such as Populus tremuloides), or Quercusrobur.

In some embodiments, the source organism is an algae, such as a greenalgae, red algae, glaucophytes, chlorarachniophytes, euglenids,chromista, or dinoflagellates.

In some embodiments, the source organism is a cyanobacteria, such ascyanobacteria classified into any of the following groups based onmorphology: Chroococcales, Pleurocapsales, Oscillatoriales, Nostocales,or Stigonematales.

In some embodiments, the source organism is an anaerobic organism.Anaerobic organisms can include, but are not limited to, obligateanaerobes, facultatitive anaerobes, and aerotolerant anaerobes. Suchorganisms can be any of the organisms listed above, bacteria, yeast,etc. In one embodiment, the obligate anaerobes can be any one orcombination selected from the group consisting of Clostridiumljungdahlii, Clostridium autoethanogenum, Eurobacterium limosum,Clostridium carboxydivorans, Peptostreptococcus productus, andButyribacterium methylotrophicum. It is to be understood that anycombination of any of the source organisms described herein can be usedfor other embodiments of the invention.

Exemplary Host Cells

A variety of host cells can be used to express isoprene synthase, DXS,IDI, MVA pathway, hydrogenase, hydrogenase maturation and/ortranscription factor polypeptides and to co-produce isoprene andhydrogen in the methods described herein. Exemplary host cells includecells from any of the organisms listed in the prior section under theheading “Exemplary Source Organisms.” The host cell may be a cell thatnaturally produces isoprene or a cell that does not naturally produceisoprene. In some embodiments, the host cell naturally produces isopreneusing the DXP pathway, and an isoprene synthase, DXS, and/or IDI nucleicacid is added to enhance production of isoprene using this pathway. Insome embodiments, the host cell naturally produces isoprene using theMVA pathway, and an isoprene synthase and/or one or more MVA pathwaynucleic acids are added to enhance production of isoprene using thispathway. In some embodiments, the host cell naturally produces isopreneusing the DXP pathway and one or more MVA pathway nucleic acids areadded to produce isoprene using part or all of the MVA pathway as wellas the DXP pathway. In some embodiments, the host cell naturallyproduces isoprene using both the DXP and MVA pathways and one or moreisoprene synthase, DXS, IDI, or MVA pathway nucleic acids are added toenhance production of isoprene by one or both of these pathways.

In some embodiments, the host cell naturally produces isoprene usingboth the DXP and MVA pathways, and one or more isoprene synthase, DXS,IDI, or MVA pathway nucleic acids are added to enhance production ofisoprene by one or both of these pathways, one or more hydrogenasenucleic acids are added to enhance hydrogen production and one or morefermentation side product-producing genes are inactivated or deleted tolimit production of fermentation side products. In some embodiments, thehost cell naturally co-produces isoprene and hydrogen using both the DXPand MVA pathways and one or more isoprene synthase, DXS, IDI, or MVApathway nucleic acids are added to enhance production of isoprene by oneor both of these pathways, one or more hydrogenase nucleic acids areadded to enhance hydrogen production, one or more fermentation sideproduct-producing genes are inactivated or deleted to limit productionof fermentation side products, and one or more hydrogen reuptake genesare inactivated or deleted to increase hydrogen production. In someembodiments, the host cell naturally co-produces isoprene and hydrogenusing both the DXP and MVA pathways and a hydrogenase, and one or moreisoprene synthase, DXS, IDI, or MVA pathway nucleic acids are added toenhance production of isoprene by one or both of these pathways, one ormore hydrogenase nucleic acids are added to enhance hydrogen production,one or more hydrogenase maturation nucleic acids are added to enhancehydrogen production, one or more fermentation side product-producinggenes are inactivated or deleted to limit production of fermentationside products, and one or more hydrogen reuptake genes are inactivatedor deleted to increase hydrogen production. In some embodiments, thehost cell naturally co-produces isoprene and hydrogen using both the DXPand MVA pathways and one or more isoprene synthase, DXS, IDI, or MVApathway nucleic acids are added to enhance production of isoprene by oneor both of these pathways, one or more hydrogenase nucleic acids areadded to enhance hydrogen production, one or more hydrogenase maturationnucleic acids are added to enhance hydrogen production, one or moretranscription factor nucleic acids are added or inactivated or deletedto enhance hydrogenase production, one or more fermentation sideproduct-producing genes are inactivated or deleted to limit productionof fermentation side products, and one or more hydrogen reuptake genesare inactivated or deleted to increase hydrogen production.

Exemplary Transformation Methods

Isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenasematuration and/or transcription factor nucleic acids or vectorscontaining them can be inserted into a host cell (e.g., a plant cell, afungal cell, a yeast cell, or a bacterial cell described herein) usingstandard techniques for expression of the encoded isoprene synthase,DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/ortranscription factor polypeptide. Introduction of a DNA construct orvector into a host cell can be performed using techniques such astransformation, electroporation, nuclear microinjection, transduction,transfection (e.g., lipofection mediated or DEAE-Dextrin mediatedtransfection or transfection using a recombinant phage virus),incubation with calcium phosphate DNA precipitate, high velocitybombardment with DNA-coated microprojectiles, and protoplast fusion.General transformation techniques are known in the art (see, e.g.,Current Protocols in Molecular Biology (F. M. Ausubel et al. (eds)Chapter 9, 1987; Sambrook et al., Molecular Cloning: A LaboratoryManual, 2^(nd) ed., Cold Spring Harbor, 1989; and Campbell et al., Curr.Genet. 16:53-56, 1989, which are each hereby incorporated by referencein their entireties, particularly with respect to transformationmethods). The expression of heterologous polypeptide in Trichoderma isdescribed in U.S. Pat. No. 6,022,725; U.S. Pat. No. 6,268,328; U.S. Pat.No. 7,262,041; WO 2005/001036; Harkki et al., Enzyme Microb. Technol.13:227-233, 1991; Harkki et al., Bio Technol. 7:596-603, 1989; EP244,234; EP 215,594; and Nevalainen et al., “The Molecular Biology ofTrichoderma and its Application to the Expression of Both Homologous andHeterologous Genes,” in Molecular Industrial Mycology, Eds. Leong andBerka, Marcel Dekker Inc., NY pp. 129-148, 1992, which are each herebyincorporated by reference in their entireties, particularly with respectto transformation and expression methods). Reference is also made to Caoet al., (Sci. 9:991-1001, 2000; EP 238023; and Yelton et al.,Proceedings. Natl. Acad. Sci. USA 81:1470-1474, 1984 (which are eachhereby incorporated by reference in their entireties, particularly withrespect to transformation methods) for transformation of Aspergillusstrains. The introduced nucleic acids may be integrated into chromosomalDNA or maintained as extrachromosomal replicating sequences.

Any method known in the art may be used to select transformants. In onenon-limiting example, stable transformants including an amdS marker aredistinguished from unstable transformants by their faster growth rateand the formation of circular colonies with a smooth, rather than raggedoutline on solid culture medium containing acetamide. Additionally, insome cases a further test of stability is conducted by growing thetransformants on a solid non-selective medium (e.g., a medium that lacksacetamide), harvesting spores from this culture medium, and determiningthe percentage of these spores which subsequently germinate and grow onselective medium containing acetamide.

In some embodiments, fungal cells are transformed by a process involvingprotoplast formation and transformation of the protoplasts followed byregeneration of the cell wall in a known manner. In one specificembodiment, the preparation of Trichoderma sp. for transformationinvolves the preparation of protoplasts from fungal mycelia (see,Campbell et al., Curr. Genet. 16:53-56, 1989, which is incorporated byreference in its entirety, particularly with respect to transformationmethods). In some embodiments, the mycelia are obtained from germinatedvegetative spores. The mycelia are treated with an enzyme that digeststhe cell wall resulting in protoplasts. The protoplasts are thenprotected by the presence of an osmotic stabilizer in the suspendingmedium. These stabilizers include sorbitol, mannitol, potassiumchloride, magnesium sulfate, and the like. Usually the concentration ofthese stabilizers varies between 0.8 M and 1.2 M. It is desirable to useabout a 1.2 M solution of sorbitol in the suspension medium.

Uptake of DNA into the host Trichoderma sp. strain is dependent upon thecalcium ion concentration. Generally, between about 10 mM CaCl₂ and 50mM CaCl₂ is used in an uptake solution. In addition to the calcium ionin the uptake solution, other compounds generally included are abuffering system such as TE buffer (10 Mm Tris, pH 7.4; 1 mM EDTA) or 10mM MOPS, pH 6.0 buffer (morpholinepropanesulfonic acid) and polyethyleneglycol (PEG). While not intending to be bound to any particular theory,it is believed that the polyethylene glycol acts to fuse the cellmembranes, thus permitting the contents of the medium to be deliveredinto the cytoplasm of the Trichoderma sp. strain and the plasmid DNA tobe transferred to the nucleus. This fusion frequently leaves multiplecopies of the plasmid DNA integrated into the host chromosome.

Usually a suspension containing the Trichoderma sp. protoplasts or cellsthat have been subjected to a permeability treatment at a density of 10⁵to 10⁷/mL (such as 2×10⁶/mL) are used in the transformation. A volume of100 μL of these protoplasts or cells in an appropriate solution (e.g.,1.2 M sorbitol and 50 mM CaCl₂) are mixed with the desired DNA.Generally, a high concentration of PEG is added to the uptake solution.From 0.1 to 1 volume of 25% PEG 4000 can be added to the protoplastsuspension. In some embodiments, about 0.25 volumes are added to theprotoplast suspension. Additives such as dimethyl sulfoxide, heparin,spermidine, potassium chloride, and the like may also be added to theuptake solution and aid in transformation. Similar procedures areavailable for other fungal host cells (see, e.g., U.S. Pat. Nos.6,022,725 and 6,268,328, which are each hereby incorporated by referencein their entireties, particularly with respect to transformationmethods).

Generally, the mixture is then cultured at approximately 0° C. for aperiod of between 10 to 30 minutes. Additional PEG is then added to themixture to further enhance the uptake of the desired nucleic acidsequence. The 25% PEG 4000 is generally added in volumes of 5 to 15times the volume of the transformation mixture; however, greater andlesser volumes may be suitable. The 25% PEG 4000 is desirably about 10times the volume of the transformation mixture. After the PEG is added,the transformation mixture is then cultured either at room temperatureor on ice before the addition of a sorbitol and CaCl₂ solution. Theprotoplast suspension is then further added to molten aliquots of agrowth medium. When the growth medium includes a growth selection (e.g.,acetamide or an antibiotic) it permits the growth of transformants only.

The transformation of bacterial cells may be performed according toconventional methods, e.g., as described in Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor, 1982, which is herebyincorporated by reference in its entirety, particularly with respect totransformation methods.

Exemplary Cell Culture Media

Also described herein is a cell or a population of cells in culture thatco-produce isoprene and hydrogen. By “cells in culture” is meant two ormore cells in a solution (e.g., a cell growth medium) that allows thecells to undergo one or more cell divisions. “Cells in culture” do notinclude plant cells that are part of a living, multicellular plantcontaining cells that have differentiated into plant tissues. In variousembodiments, the cell culture includes at least or about 10, 20, 50,100, 200, 500, 1,000, 5,000, 10,000 or more cells.

By “cells in oxygen-limited culture” is meant two or more cells in asolution (e.g., a cell growth medium) that allows the cell to under goone or more cell divisions, wherein the solution contains a limitingamount of oxygen. The term “oxygen-limited culture” means that theculture is either anoxic or contains less than the required amount ofoxygen to support respiration via the biological transfer of reducingequivalents to oxygen, and also encompasses anaerobic cultures. Underoxygen-limited culture conditions, some electrons derived from carbonmetabolism cannot be accepted because oxygen concentrations are too low,causing cells to switch to hydrogen production if they comprise theappropriate metabolic pathways for doing so. Oxygen-limited cultureconditions occur when the oxygen transfer rate (“OTR”) is less than theoxygen uptake rate (“OUR”) indicated by dissolved oxygen concentrationsof close to zero in culture medium.

Any carbon source can be used to cultivate the host cells. The term“carbon source” refers to one or more carbon-containing compoundscapable of being metabolized by a host cell or organism. For example,the cell medium used to cultivate the host cells may include any carbonsource suitable for maintaining the viability or growing the host cells.

In some embodiments, the carbon source is a carbohydrate (such asmonosaccharide, disaccharide, oligosaccharide, or polysaccharides),invert sugar (e.g., enzymatically treated sucrose syrup), glycerol,glycerine (e.g., a glycerine byproduct of a biodiesel or soap-makingprocess), dihydroxyacetone, one-carbon source, oil (e.g., a plant orvegetable oil such as corn, palm, or soybean oil), animal fat, animaloil, fatty acid (e.g., a saturated fatty acid, unsaturated fatty acid,or polyunsaturated fatty acid), lipid, phospholipid, glycerolipid,monoglyceride, diglyceride, triglyceride, polypeptide (e.g., a microbialor plant protein or peptide), renewable carbon source (e.g., a biomasscarbon source such as a hydrolyzed biomass carbon source), yeastextract, component from a yeast extract, polymer, acid, alcohol,aldehyde, ketone, amino acid, succinate, lactate, acetate, ethanol, orany combination of two or more of the foregoing. In some embodiments,the carbon source is a product of photosynthesis, including, but notlimited to, glucose.

Exemplary monosaccharides include glucose and fructose; exemplaryoligosaccharides include lactose and sucrose, and exemplarypolysaccharides include starch and cellulose. Exemplary carbohydratesinclude C6 sugars (e.g., fructose, mannose, galactose, or glucose) andC5 sugars (e.g., xylose or arabinose). In some embodiments, the cellmedium includes a carbohydrate as well as a carbon source other than acarbohydrate (e.g., glycerol, glycerine, dihydroxyacetone, one-carbonsource, oil, animal fat, animal oil, fatty acid, lipid, phospholipid,glycerolipid, monoglyceride, diglyceride, triglyceride, renewable carbonsource, or a component from a yeast extract). In some embodiments, thecell medium includes a carbohydrate as well as a polypeptide (e.g., amicrobial or plant protein or peptide). In some embodiments, themicrobial polypeptide is a polypeptide from yeast or bacteria. In someembodiments, the plant polypeptide is a polypeptide from soy, corn,canola, jatropha, palm, peanut, sunflower, coconut, mustard, rapeseed,cottonseed, palm kernel, olive, safflower, sesame, or linseed.

In some embodiments, the concentration of the carbohydrate is at leastor about 5 grams per liter of broth (g/L, wherein the volume of brothincludes both the volume of the cell medium and the volume of thecells), such as at least or about 10, 15, 20, 30, 40, 50, 60, 80, 100,150, 200, 300, 400, or more g/L. In some embodiments, the concentrationof the carbohydrate is between about 50 and about 400 g/L, such asbetween about 100 and about 360 g/L, between about 120 and about 360g/L, or between about 200 and about 300 g/L. In some embodiments, thisconcentration of carbohydrate includes the total amount of carbohydratethat is added before and/or during the culturing of the host cells.

In some embodiments, the cells are cultured under limited glucoseconditions. By “limited glucose conditions” is meant that the amount ofglucose that is added is less than or about 105% (such as about 100%) ofthe amount of glucose that is consumed by the cells. In particularembodiments, the amount of glucose that is added to the culture mediumis approximately the same as the amount of glucose that is consumed bythe cells during a specific period of time. In some embodiments, therate of cell growth is controlled by limiting the amount of addedglucose such that the cells grow at the rate that can be supported bythe amount of glucose in the cell medium. In some embodiments, glucosedoes not accumulate during the time the cells are cultured. In variousembodiments, the cells are cultured under limited glucose conditions forgreater than or about 1, 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, or70 hours. In various embodiments, the cells are cultured under limitedglucose conditions for greater than or about 5, 10, 15, 20, 25, 30, 35,40, 50, 60, 70, 80, 90, 95, or 100% of the total length of time thecells are cultured. While not intending to be bound by any particulartheory, it is believed that limited glucose conditions may allow morefavorable regulation of the cells.

In some embodiments, the cells are cultured in the presence of an excessof glucose. In particular embodiments, the amount of glucose that isadded is greater than about 105% (such as about or greater than 110,120, 150, 175, 200, 250, 300, 400, or 500%) or more of the amount ofglucose that is consumed by the cells during a specific period of time.In some embodiments, glucose accumulates during the time the cells arecultured.

Exemplary lipids are any substance containing one or more fatty acidsthat are C4 and above fatty acids that are saturated, unsaturated, orbranched.

Exemplary oils are lipids that are liquid at room temperature. In someembodiments, the lipid contains one or more C4 or above fatty acids(e.g., contains one or more saturated, unsaturated, or branched fattyacid with four or more carbons). In some embodiments, the oil isobtained from soy, corn, canola, jatropha, palm, peanut, sunflower,coconut, mustard, rapeseed, cottonseed, palm kernel, olive, safflower,sesame, linseed, oleagineous microbial cells, Chinese tallow, or anycombination of two or more of the foregoing.

Exemplary fatty acids include compounds of the formula RCOOH, where “R”is a hydrocarbon. Exemplary unsaturated fatty acids include compoundswhere “R” includes at least one carbon-carbon double bond. Exemplaryunsaturated fatty acids include, but are not limited to, oleic acid,vaccenic acid, linoleic acid, palmitoleic acid, and arachidonic acid.Exemplary polyunsaturated fatty acids include compounds where “R”includes a plurality of carbon-carbon double bonds. Exemplary saturatedfatty acids include compounds where “R” is a saturated aliphatic group.In some embodiments, the carbon source includes one or more C₁₂-C₂₂fatty acids, such as a C₁₂ saturated fatty acid, a C₁₄ saturated fattyacid, a C₁₆ saturated fatty acid, a C₁₈ saturated fatty acid, a C₂₀saturated fatty acid, or a C₂₂ saturated fatty acid. In an exemplaryembodiment, the fatty acid is palmitic acid. In some embodiments, thecarbon source is a salt of a fatty acid (e.g., an unsaturated fattyacid), a derivative of a fatty acid (e.g., an unsaturated fatty acid),or a salt of a derivative of fatty acid (e.g., an unsaturated fattyacid). Suitable salts include, but are not limited to, lithium salts,potassium salts, sodium salts, and the like. Di- and triglycerols arefatty acid esters of glycerol.

In some embodiments, the concentration of the lipid, oil, fat, fattyacid, monoglyceride, diglyceride, or triglyceride is at least or about 1gram per liter of broth (g/L, wherein the volume of broth includes boththe volume of the cell medium and the volume of the cells), such as atleast or about 5, 10, 15, 20, 30, 40, 50, 60, 80, 100, 150, 200, 300,400, or more g/L. In some embodiments, the concentration of the lipid,oil, fat, fatty acid, monoglyceride, diglyceride, or triglyceride isbetween about 10 and about 400 g/L, such as between about 25 and about300 g/L, between about 60 and about 180 g/L, or between about 75 andabout 150 g/L. In some embodiments, the concentration includes the totalamount of the lipid, oil, fat, fatty acid, monoglyceride, diglyceride,or triglyceride that is added before and/or during the culturing of thehost cells. In some embodiments, the carbon source includes both (i) alipid, oil, fat, fatty acid, monoglyceride, diglyceride, or triglycerideand (ii) a carbohydrate, such as glucose. In some embodiments, the ratioof the lipid, oil, fat, fatty acid, monoglyceride, diglyceride, ortriglyceride to the carbohydrate is about 1:1 on a carbon basis (i.e.,one carbon in the lipid, oil, fat, fatty acid, monoglyceride,diglyceride, or triglyceride per carbohydrate carbon). In particularembodiments, the amount of the lipid, oil, fat, fatty acid,monoglyceride, diglyceride, or triglyceride is between about 60 and 180g/L, and the amount of the carbohydrate is between about 120 and 360g/L.

Exemplary microbial polypeptide carbon sources include one or morepolypeptides from yeast or bacteria. Exemplary plant polypeptide carbonsources include one or more polypeptides from soy, corn, canola,jatropha, palm, peanut, sunflower, coconut, mustard, rapeseed,cottonseed, palm kernel, olive, safflower, sesame, or linseed.

Exemplary renewable carbon sources include cheese whey permeate,cornsteep liquor, sugar beet molasses, barley malt, and components fromany of the foregoing. Exemplary renewable carbon sources also includeglucose, hexose, pentose and xylose present in biomass, such as corn,switchgrass, sugar cane, cell waste of fermentation processes, andprotein by-product from the milling of soy, corn, or wheat. In someembodiments, the biomass carbon source is a lignocellulosic,hemicellulosic, or cellulosic material such as, but are not limited to,a grass, wheat, wheat straw, bagasse, sugar cane bagasse, soft woodpulp, corn, corn cob or husk, corn kernel, fiber from corn kernels, cornstover, switch grass, rice hull product, or a by-product from wet or drymilling of grains (e.g., corn, sorghum, rye, triticate, barley, wheat,and/or distillers grains). Exemplary cellulosic materials include wood,paper and pulp waste, herbaceous plants, and fruit pulp. In someembodiments, the carbon source includes any plant part, such as stems,grains, roots, or tubers. In some embodiments, all or part of any of thefollowing plants are used as a carbon source: corn, wheat, rye, sorghum,triticate, rice, millet, barley, cassaya, legumes, such as beans andpeas, potatoes, sweet potatoes, bananas, sugarcane, and/or tapioca. Insome embodiments, the carbon source is a biomass hydrolysate, such as abiomass hydrolysate that includes both xylose and glucose or thatincludes both sucrose and glucose.

In some embodiments, the renewable carbon source (such as biomass) ispretreated before it is added to the cell culture medium. In someembodiments, the pretreatment includes enzymatic pretreatment, chemicalpretreatment, or a combination of both enzymatic and chemicalpretreatment (see, for example, Farzaneh et al., Bioresource Technology96 (18): 2014-2018, 2005; U.S. Pat. No. 6,176,176; U.S. Pat. No.6,106,888; which are each hereby incorporated by reference in theirentireties, particularly with respect to the pretreatment of renewablecarbon sources). In some embodiments, the renewable carbon source ispartially or completely hydrolyzed before it is added to the cellculture medium.

In some embodiments, the renewable carbon source (such as corn stover)undergoes ammonia fiber expansion (AFEX) pretreatment before it is addedto the cell culture medium (see, for example, Farzaneh et al.,Bioresource Technology 96 (18): 2014-2018, 2005). During AFEXpretreatment, a renewable carbon source is treated with liquid anhydrousammonia at moderate temperatures (such as about 60 to about 100° C.) andhigh pressure (such as about 250 to about 300 psi) for about 5 minutes.Then, the pressure is rapidly released. In this process, the combinedchemical and physical effects of lignin solubilization, hemicellulosehydrolysis, cellulose decrystallization, and increased surface areaenables near complete enzymatic conversion of cellulose andhemicellulose to fermentable sugars. AFEX pretreatment has the advantagethat nearly all of the ammonia can be recovered and reused, while theremaining serves as nitrogen source for microbes in downstreamprocesses. Also, a wash stream is not required for AFEX pretreatment.Thus, dry matter recovery following the AFEX treatment is essentially100%. AFEX is basically a dry to dry process. The treated renewablecarbon source is stable for long periods and can be fed at very highsolid loadings in enzymatic hydrolysis or fermentation processes.Cellulose and hemicellulose are well preserved in the AFEX process, withlittle or no degradation. There is no need for neutralization prior tothe enzymatic hydrolysis of a renewable carbon source that has undergoneAFEX pretreatment. Enzymatic hydrolysis of AFEX-treated carbon sourcesproduces clean sugar streams for subsequent fermentation use.

In some embodiments, the concentration of the carbon source (e.g., arenewable carbon source) is equivalent to at least or about 0.1, 0.5, 1,1.5 2, 3, 4, 5, 10, 15, 20, 30, 40, or 50% glucose (w/v). The equivalentamount of glucose can be determined by using standard HPLC methods withglucose as a reference to measure the amount of glucose generated fromthe carbon source. In some embodiments, the concentration of the carbonsource (e.g., a renewable carbon source) is equivalent to between about0.1 and about 20% glucose, such as between about 0.1 and about 10%glucose, between about 0.5 and about 10% glucose, between about 1 andabout 10% glucose, between about 1 and about 5% glucose, or betweenabout 1 and about 2% glucose.

In some embodiments, the carbon source includes yeast extract or one ormore components of yeast extract. In some embodiments, the concentrationof yeast extract is at least 1 gram of yeast extract per liter of broth(g/L, wherein the volume of broth includes both the volume of the cellmedium and the volume of the cells), such at least or about 5, 10, 15,20, 30, 40, 50, 60, 80, 100, 150, 200, 300, or more g/L. In someembodiments, the concentration of yeast extract is between about 1 andabout 300 g/L, such as between about 1 and about 200 g/L, between about5 and about 200 g/L, between about 5 and about 100 g/L, or between about5 and about 60 g/L. In some embodiments, the concentration includes thetotal amount of yeast extract that is added before and/or during theculturing of the host cells. In some embodiments, the carbon sourceincludes both yeast extract (or one or more components thereof) andanother carbon source, such as glucose. In some embodiments, the ratioof yeast extract to the other carbon source is about 1:5, about 1:10, orabout 1:20 (w/w).

Additionally the carbon source may also be one-carbon substrates such ascarbon dioxide, or methanol. Glycerol production from single carbonsources (e.g., methanol, formaldehyde, or formate) has been reported inmethylotrophic yeasts (Yamada et al., Agric. Biol. Chem., 53(2) 541-543,1989, which is hereby incorporated by reference in its entirety,particularly with respect to carbon sources) and in bacteria (Hunter et.al., Biochemistry, 24, 4148-4155, 1985, which is hereby incorporated byreference in its entirety, particularly with respect to carbon sources).These organisms can assimilate single carbon compounds, ranging inoxidation state from methane to formate, and produce glycerol. Thepathway of carbon assimilation can be through ribulose monophosphate,through serine, or through xylulose-momophosphate (Gottschalk, BacterialMetabolism, Second Edition, Springer-Verlag: New York, 1986, which ishereby incorporated by reference in its entirety, particularly withrespect to carbon sources). The ribulose monophosphate pathway involvesthe condensation of formate with ribulose-5-phosphate to form a sixcarbon sugar that becomes fructose and eventually the three carbonproduct glyceraldehyde-3-phosphate. Likewise, the serine pathwayassimilates the one-carbon compound into the glycolytic pathway viamethylenetetrahydrofolate.

In addition to one and two carbon substrates, methylotrophic organismsare also known to utilize a number of other carbon containing compoundssuch as methylamine, glucosamine and a variety of amino acids formetabolic activity. For example, methylotrophic yeast are known toutilize the carbon from methylamine to form trehalose or glycerol(Bellion et al., Microb. Growth Cl Compd., [Int. Symp.], 7^(th) ed.,415-32. Editors: Murrell et al., Publisher: Intercept, Andover, UK,1993, which is hereby incorporated by reference in its entirety,particularly with respect to carbon sources). Similarly, various speciesof Candida metabolize alanine or oleic acid (Sulter et al., Arch.Microbiol. 153(5), 485-9, 1990, which is hereby incorporated byreference in its entirety, particularly with respect to carbon sources).

In some embodiments, cells are cultured in a standard medium containingphysiological salts and nutrients (see, e.g., Pourquie, J. et al.,Biochemistry and Genetics of Cellulose Degradation, eds. Aubert et al.,Academic Press, pp. 71-86, 1988 and Ilmen et al., Appl. Environ.Microbiol. 63:1298-1306, 1997, which are each hereby incorporated byreference in their entireties, particularly with respect to cellmedias). Exemplary growth media are common commercially prepared mediasuch as Luria Bertani (LB) broth, Sabouraud Dextrose (SD) broth, orYeast medium (YM) broth. Other defined or synthetic growth media mayalso be used, and the appropriate medium for growth of particular hostcells are known by someone skilled in the art of microbiology orfermentation science.

In addition to an appropriate carbon source, the cell medium desirablycontains suitable minerals, salts, cofactors, buffers, and othercomponents known to those skilled in the art suitable for the growth ofthe cultures or the enhancement of isoprene production (see, forexample, WO 2004/033646 and references cited therein and WO 96/35796 andreferences cited therein, which are each hereby incorporated byreference in their entireties, particularly with respect cell medias andcell culture conditions). In some embodiments where an isoprenesynthase, DXS, IDI, and/or MVA pathway nucleic acid is under the controlof an inducible promoter, the inducing agent (e.g., a sugar, metal saltor antimicrobial), is desirably added to the medium at a concentrationeffective to induce expression of an isoprene synthase, DXS, IDI, and/orMVA pathway polypeptide. In some embodiments, cell medium has anantibiotic (such as kanamycin) that corresponds to the antibioticresistance nucleic acid (such as a kanamycin resistance nucleic acid) ona vector that has one or more DXS, IDI, or MVA pathway nucleic acids.

Exemplary Cell Culture Conditions

Materials and methods suitable for the maintenance and growth ofbacterial cultures are well known in the art. Exemplary techniques maybe found in Manual of Methods for General Bacteriology Gerhardt et al.,eds), American Society for Microbiology, Washington, D.C. (1994) orBrock in Biotechnology: A Textbook of Industrial Microbiology, SecondEdition (1989) Sinauer Associates, Inc., Sunderland, Mass., which areeach hereby incorporated by reference in their entireties, particularlywith respect to cell culture techniques. In some embodiments, the cellsare cultured in a culture medium under conditions permitting theexpression of one or more isoprene synthase, DXS, IDI, or MVA pathwaypolypeptides encoded by a nucleic acid inserted into the host cells.

Standard cell culture conditions can be used to culture the cells (see,for example, WO 2004/033646 and references cited therein, which are eachhereby incorporated by reference in their entireties, particularly withrespect to cell culture and fermentation conditions). Cells are grownand maintained at an appropriate temperature, gas mixture, and pH (suchas at about 20° C. to about 37° C., at about 6% to about 84% CO₂, and ata pH between about 5 to about 9). In some embodiments, cells are grownat 35° C. in an appropriate cell medium. In some embodiments, e.g.,cultures are cultured at approximately 28° C. in appropriate medium inshake cultures or fermentors until the desired amount of isoprene andhydrogen co-production is achieved. In some embodiments, the pH rangesfor fermentation are between about pH 5.0 to about pH 9.0 (such as aboutpH 6.0 to about pH 8.0 or about 6.5 to about 7.0). Reactions may beperformed under aerobic, anoxic, or anaerobic conditions based on therequirements of the host cells. In some embodiments, the cells arecultured under oxygen-limited conditions. In some embodiments, the cellsare cultured in the presence of oxygen under conditions where 0.5 molesof oxygen are taken up per mole of isoprene produced. In someembodiments, the cells are cultured under anaerobic conditions.Exemplary culture conditions for a given filamentous fungus are known inthe art and may be found in the scientific literature and/or from thesource of the fungi such as the American Type Culture Collection andFungal Genetics Stock Center.

In various embodiments, the cells are grown using any known mode offermentation, such as batch, fed-batch, or continuous processes. In someembodiments, a batch method of fermentation is used. Classical batchfermentation is a closed system where the composition of the media isset at the beginning of the fermentation and is not subject toartificial alterations during the fermentation. Thus, at the beginningof the fermentation the cell medium is inoculated with the desired hostcells and fermentation is permitted to occur adding nothing to thesystem. Typically, however, “batch” fermentation is batch with respectto the addition of carbon source and attempts are often made atcontrolling factors such as pH and oxygen concentration. In batchsystems, the metabolite and biomass compositions of the system changeconstantly until the time the fermentation is stopped. Within batchcultures, cells moderate through a static lag phase to a high growth logphase and finally to a stationary phase where growth rate is diminishedor halted. In some embodiments, cells in log phase are responsible forthe bulk of the isoprene production. In some embodiments, cells instationary phase produce isoprene.

In some embodiments, a variation on the standard batch system is used,such as the Fed-Batch system. Fed-Batch fermentation processes comprisea typical batch system with the exception that the carbon source isadded in increments as the fermentation progresses. Fed-Batch systemsare useful when catabolite repression is apt to inhibit the metabolismof the cells and where it is desirable to have limited amounts of carbonsource in the cell medium. Fed-batch fermentations may be performed withthe carbon source (e.g., glucose) in a limited or excess amount.Measurement of the actual carbon source concentration in Fed-Batchsystems is difficult and is therefore estimated on the basis of thechanges of measurable factors such as pH, dissolved oxygen, and thepartial pressure of waste gases such as CO₂. Batch and Fed-Batchfermentations are common and well known in the art and examples may befound in Brock, Biotechnology: A Textbook of Industrial Microbiology,Second Edition (1989) Sinauer Associates, Inc., which is herebyincorporated by reference in its entirety, particularly with respect tocell culture and fermentation conditions.

In some embodiments, continuous fermentation methods are used.Continuous fermentation is an open system where a defined fermentationmedium is added continuously to a bioreactor and an equal amount ofconditioned medium is removed simultaneously for processing. Continuousfermentation generally maintains the cultures at a constant high densitywhere cells are primarily in log phase growth.

Continuous fermentation allows for the modulation of one factor or anynumber of factors that affect cell growth or isoprene production. Forexample, one method maintains a limiting nutrient such as the carbonsource or nitrogen level at a fixed rate and allows all other parametersto moderate. In other systems, a number of factors affecting growth canbe altered continuously while the cell concentration (e.g., theconcentration measured by media turbidity) is kept constant. Continuoussystems strive to maintain steady state growth conditions. Thus, thecell loss due to media being drawn off is balanced against the cellgrowth rate in the fermentation. Methods of modulating nutrients andgrowth factors for continuous fermentation processes as well astechniques for maximizing the rate of product formation are well knownin the art of industrial microbiology and a variety of methods aredetailed by Brock, Biotechnology: A Textbook of Industrial Microbiology,Second Edition (1989) Sinauer Associates, Inc., which is herebyincorporated by reference in its entirety, particularly with respect tocell culture and fermentation conditions.

In some embodiments, cells are immobilized on a substrate as whole cellcatalysts and subjected to fermentation conditions for isopreneproduction.

In some embodiments, bottles of liquid culture are placed in shakers inorder to introduce oxygen to the liquid and maintain the uniformity ofthe culture. In some embodiments, an incubator is used to control thetemperature, humidity, shake speed, and/or other conditions in which aculture is grown. The simplest incubators are insulated boxes with anadjustable heater, typically going up to ˜65° C. More elaborateincubators can also include the ability to lower the temperature (viarefrigeration), or the ability to control humidity or CO₂ levels. Mostincubators include a timer; some can also be programmed to cycle throughdifferent temperatures, humidity levels, etc. Incubators can vary insize from tabletop to units the size of small rooms.

If desired, a portion or all of the cell medium can be changed toreplenish nutrients and/or avoid the build up of potentially harmfulmetabolic byproducts and dead cells. In the case of suspension cultures,cells can be separated from the media by centrifuging or filtering thesuspension culture and then resuspending the cells in fresh media. Inthe case of adherent cultures, the media can be removed directly byaspiration and replaced. In some embodiments, the cell medium allows atleast a portion of the cells to divide for at least or about 5, 10, 20,40, 50, 60, 65, or more cell divisions in a continuous culture (such asa continuous culture without dilution).

In some embodiments, a constitutive or leaky promoter (such as a Trcpromoter) is used and a compound (such as IPTG) is not added to induceexpression of the isoprene synthase, DXS, IDI, or MVA pathway nucleicacid(s) operably linked to the promoter. In some embodiments, a compound(such as IPTG) is added to induce expression of the isoprene synthase,DXS, IDI, or MVA pathway nucleic acid(s) operably linked to thepromoter.

Exemplary Methods for Decoupling Isoprene Production from Cell Growth

Desirably, carbon from the feedstock is converted to isoprene ratherthan to the growth and maintenance of the cells. In some embodiments,the cells are grown to a low to medium OD₆₀₀, then production ofisoprene is started or increased. This strategy permits a large portionof the carbon to be converted to isoprene.

In some embodiments, cells reach an optical density such that they nolonger divide or divide extremely slowly, but continue to make isoprenefor several hours (such as about 2, 4, 6, 8, 10, 15, 20, 25, 30, or morehours). For example, FIGS. 60A-67C illustrate that cells may continue toproduce a substantial amount of mevalonic acid or isoprene after thecells reach an optical density such that they no longer divide or divideextremely slowly. In some cases, the optical density at 550 nm decreasesover time (such as a decrease in the optical density after the cells areno longer in an exponential growth phase due to cell lysis), and thecells continue to produce a substantial amount of mevalonic acid orisoprene. In some embodiments, the optical density at 550 nm of thecells increases by less than or about 50% (such as by less than or about40, 30, 20, 10, 5, or 0%) over a certain time period (such as greaterthan or about 5, 10, 15, 20, 25, 30, 40, 50 or 60 hours), and the cellsproduce isoprene at greater than or about 1, 10, 25, 50, 100, 150, 200,250, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750,2,000, 2,500, 3,000, 4,000, 5,000, or more nmole of isoprene/gram ofcells for the wet weight of the cells/hour (nmole/g_(wcm)/hr) duringthis time period. In some embodiments, the amount of isoprene is betweenabout 2 to about 5,000 nmole/g_(wcm)/hr, such as between about 2 toabout 100 nmole/g_(wcm)/hr, about 100 to about 500 nmole/g_(wcm)/hr,about 150 to about 500 nmole/g_(wcm)/hr, about 500 to about 1,000nmole/g_(wcm)/hr, about 1,000 to about 2,000 nmole/g_(wcm)/hr, or about2,000 to about 5,000 nmole/g_(wcm)/hr. In some embodiments, the amountof isoprene is between about 20 to about 5,000 nmole/g_(wcm)/hr, about100 to about 5,000 nmole/g_(wcm)/hr, about 200 to about 2,000nmole/g_(wcm)/hr, about 200 to about 1,000 nmole/g_(wcm)/hr, about 300to about 1,000 nmole/g_(wcm)/hr, or about 400 to about 1,000nmole/g_(wcm)/hr.

In some embodiments, the optical density at 550 nm of the cellsincreases by less than or about 50% (such as by less than or about 40,30, 20, 10, 5, or 0%) over a certain time period (such as greater thanor about 5, 10, 15, 20, 25, 30, 40, 50 or 60 hours), and the cellsproduce a cumulative titer (total amount) of isoprene at greater than orabout 1, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800,900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000,10,000, 50,000, 100,000, or more mg of isoprene/L of broth(mg/L_(broth), wherein the volume of broth includes the volume of thecells and the cell medium) during this time period. In some embodiments,the amount of isoprene is between about 2 to about 5,000 mg/L_(broth),such as between about 2 to about 100 mg/L_(broth), about 100 to about500 mg/L_(broth), about 500 to about 1,000 mg/L_(broth), about 1,000 toabout 2,000 mg/L_(broth), or about 2,000 to about 5,000 mg/L_(broth). Insome embodiments, the amount of isoprene is between about 20 to about5,000 mg/L_(broth), about 100 to about 5,000 mg/L_(broth), about 200 toabout 2,000 mg/L_(broth), about 200 to about 1,000 mg/L_(broth), about300 to about 1,000 mg/L_(broth), or about 400 to about 1,000mg/L_(broth).

In some embodiments, the optical density at 550 nm of the cellsincreases by less than or about 50% (such as by less than or about 40,30, 20, 10, 5, or 0%) over a certain time period (such as greater thanor about 5, 10, 15, 20, 25, 30, 40, 50 or 60 hours), and the cellsconvert greater than or about 0.0015, 0.002, 0.005, 0.01, 0.02, 0.05,0.1, 0.12, 0.14, 0.16, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2,1.4, 1.6, 1.8, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 6.0, 7.0, or 8.0% of thecarbon in the cell culture medium into isoprene during this time period.In some embodiments, the percent conversion of carbon into isoprene isbetween such as about 0.002 to about 4.0%, about 0.002 to about 3.0%,about 0.002 to about 2.0%, about 0.002 to about 1.6%, about 0.002 toabout 0.005%, about 0.005 to about 0.01%, about 0.01 to about 0.05%,about 0.05 to about 0.15%, 0.15 to about 0.2%, about 0.2 to about 0.3%,about 0.3 to about 0.5%, about 0.5 to about 0.8%, about 0.8 to about1.0%, or about 1.0 to about 1.6%. In some embodiments, the percentconversion of carbon into isoprene is between about 0.002 to about 0.4%,0.002 to about 0.16%, 0.04 to about 0.16%, about 0.005 to about 0.3%,about 0.01 to about 0.3%, or about 0.05 to about 0.3%.

In some embodiments, isoprene is only produced in stationary phase. Insome embodiments, isoprene is produced in both the growth phase andstationary phase. In various embodiments, the amount of isopreneproduced (such as the total amount of isoprene produced or the amount ofisoprene produced per liter of broth per hour per OD₆₀₀) duringstationary phase is greater than or about 2, 3, 4, 5, 10, 20, 30, 40,50, or more times the amount of isoprene produced during the growthphase for the same length of time. In various embodiments, greater thanor about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99% or more of thetotal amount of isoprene that is produced (such as the production ofisoprene during a fermentation for a certain amount of time, such as 20hours) is produced while the cells are in stationary phase. In variousembodiments, greater than or about 5, 10, 20, 30, 40, 50, 60, 70, 80,90, 95, 99% or more of the total amount of isoprene that is produced(such as the production of isoprene during a fermentation for a certainamount of time, such as 20 hours) is produced while the cells divideslowly or not at all such that the optical density at 550 nm of thecells increases by less than or about 50% (such as by less than or about40, 30, 20, 10, 5, or 0%). In some embodiments, isoprene is onlyproduced in the growth phase.

In some embodiments, one or more MVA pathway, IDI, DXP, or isoprenesynthase nucleic acids are placed under the control of a promoter orfactor that is more active in stationary phase than in the growth phase.For example, one or more MVA pathway, IDI, DXP, or isoprene synthasenucleic acids may be placed under control of a stationary phase sigmafactor, such as RpoS. In some embodiments, one or more MVA pathway, IDI,DXP, or isoprene synthase nucleic acids are placed under control of apromoter inducible in stationary phase, such as a promoter inducible bya response regulator active in stationary phase.

Production of Isoprene Within Safe Operating Ranges

The production of isoprene within safe operating levels according to itsflammability characteristics simplifies the design and construction ofcommercial facilities, vastly improves the ability to operate safely,and limits the potential for fires to occur. In particular, the optimalranges for the production of isoprene are within the safe zone, i.e.,the nonflammable range of isoprene concentrations. In one such aspect,described herein is a method for the production of isoprene within thenonflammable range of isoprene concentrations (outside the flammabilityenvelope of isoprene).

Thus, computer modeling and experimental testing were used to determinethe flammability limits of isoprene (such as isoprene in the presence ofO₂, N₂, CO₂, or any combination of two or more of the foregoing gases)in order to ensure process safety. The flammability envelope ischaracterized by the lower flammability limit (LFL), the upperflammability limit (UFL), the limiting oxygen concentration (LOC), andthe limiting temperature. For a system to be flammable, a minimum amountof fuel (such as isoprene) must be in the presence of a minimum amountof oxidant, typically oxygen. The LFL is the minimum amount of isoprenethat must be present to sustain burning, while the UFL is the maximumamount of isoprene that can be present. Above this limit, the mixture isfuel rich and the fraction of oxygen is too low to have a flammablemixture. The LOC indicates the minimum fraction of oxygen that must alsobe present to have a flammable mixture. The limiting temperature isbased on the flash point of isoprene and is that lowest temperature atwhich combustion of isoprene can propagate. These limits are specific tothe concentration of isoprene, type and concentration of oxidant, inertspresent in the system, temperature, and pressure of the system.Compositions that fall within the limits of the flammability envelopepropagate combustion and require additional safety precautions in boththe design and operation of process equipment.

The following conditions were tested using computer simulation andmathematical analysis and experimental testing. If desired, otherconditions (such as other temperature, pressure, and permanent gascompositions) may be tested using the methods described herein todetermine the LFL, UFL, and LOC concentrations.

(1) Computer Simulation and Mathematical Analysis Test Suite 1:

isoprene: 0 wt %-14 wt %

O₂: 6 wt %-21 wt % N₂: 79 wt %-94 wt % Test Suite 2:

isoprene: 0 wt %-14 wt %

O₂: 6 wt %-21 wt % N₂: 79 wt %-94 wt %

Saturated with H₂O

Test Suite 3:

isoprene: 0 wt %-14 wt %

O₂: 6 wt %-21 wt % N₂: 79 wt %-94 wt % CO₂: 5 wt %-30 wt %

(2) Experimental testing for final determination of flammability limits

Test Suite 1:

isoprene: 0 wt %-14 wt %

O₂: 6 wt %-21 wt % N₂: 79 wt %-94 wt % Test Suite 2:

isoprene: 0 wt %-14 wt %

O₂: 6 wt %-21 wt % N₂: 79 wt %-94 wt %

Saturated with H₂O

Simulation software was used to give an estimate of the flammabilitycharacteristics of the system for several different testing conditions.CO₂ showed no significant affect on the system's flammability limits.Test suites 1 and 2 were confirmed by experimental testing. The modelingresults were in-line with the experimental test results. Only slightvariations were found with the addition of water.

The LOC was determined to be 9.5 vol % for an isoprene, O₂, N₂, and CO₂mixture at 40° C. and 1 atmosphere. The addition of up to 30% CO₂ didnot significantly affect the flammability characteristics of anisoprene, O₂, and N₂ mixture. Only slight variations in flammabilitycharacteristics were shown between a dry and water saturated isoprene,O₂, and N₂ system. The limiting temperature is about −54° C.Temperatures below about −54° C. are too low to propagate combustion ofisoprene.

In some embodiments, the LFL of isoprene ranges from about 1.5 vol. % toabout 2.0 vol %, and the UFL of isoprene ranges from about 2.0 vol. % toabout 12.0 vol. %, depending on the amount of oxygen in the system. Insome embodiments, the LOC is about 9.5 vol % oxygen. In someembodiments, the LFL of isoprene is between about 1.5 vol. % to about2.0 vol %, the UFL of isoprene is between about 2.0 vol. % to about 12.0vol. %, and the LOC is about 9.5 vol % oxygen when the temperature isbetween about 25° C. to about 55° C. (such as about 40° C.) and thepressure is between about 1 atmosphere and 3 atmospheres.

In some embodiments, isoprene is produced in the presence of less thanabout 9.5 vol % oxygen (that is, below the LOC required to have aflammable mixture of isoprene). In some embodiments in which isoprene isproduced in the presence of greater than or about 9.5 vol % oxygen, theisoprene concentration is below the LFL (such as below about 1.5 vol.%). For example, the amount of isoprene can be kept below the LFL bydiluting the isoprene composition with an inert gas (e.g., bycontinuously or periodically adding an inert gas such as nitrogen tokeep the isoprene composition below the LFL). In some embodiments inwhich isoprene is produced in the presence of greater than or about 9.5vol % oxygen, the isoprene concentration is above the UFL (such as aboveabout 12 vol. %). For example, the amount of isoprene can be kept abovethe UFL by using a system (such as any of the cell culture systemsdescribed herein) that produces isoprene at a concentration above theUFL. If desired, a relatively low level of oxygen can be used so thatthe UFL is also relatively low. In this case, a lower isopreneconcentration is needed to remain above the UFL.

In some embodiments in which isoprene is produced in the presence ofgreater than or about 9.5 vol % oxygen, the isoprene concentration iswithin the flammability envelope (such as between the LFL and the UFL).In some embodiments when the isoprene concentration may fall within theflammability envelope, one or more steps are performed to reduce theprobability of a fire or explosion. For example, one or more sources ofignition (such as any materials that may generate a spark) can beavoided. In some embodiments, one or more steps are performed to reducethe amount of time that the concentration of isoprene remains within theflammability envelope. In some embodiments, a sensor is used to detectwhen the concentration of isoprene is close to or within theflammability envelope. If desired, the concentration of isoprene can bemeasured at one or more time points during the culturing of cells, andthe cell culture conditions and/or the amount of inert gas can beadjusted using standard methods if the concentration of isoprene isclose to or within the flammability envelope. In particular embodiments,the cell culture conditions (such as fermentation conditions) areadjusted to either decrease the concentration of isoprene below the LFLor increase the concentration of isoprene above the UFL. In someembodiments, the amount of isoprene is kept below the LFL by dilutingthe isoprene composition with an inert gas (such as by continuously orperiodically adding an inert gas to keep the isoprene composition belowthe LFL).

In some embodiments, the amount of flammable volatiles other thanisoprene (such as one or more sugars) is at least about 2, 5, 10, 50,75, or 100-fold less than the amount of isoprene produced. In someembodiments, the portion of the gas phase other than isoprene gascomprises between about 0% to about 100% (volume) oxygen, such asbetween about 0% to about 10%, about 10% to about 20%, about 20% toabout 30%, about 30% to about 40%, about 40% to about 50%, about 50% toabout 60%, about 60% to about 70%, about 70% to about 80%, about 90% toabout 90%, or about 90% to about 100% (volume) oxygen. In someembodiments, the portion of the gas phase other than isoprene gascomprises between about 0% to about 99% (volume) nitrogen, such asbetween about 0% to about 10%, about 10% to about 20%, about 20% toabout 30%, about 30% to about 40%, about 40% to about 50%, about 50% toabout 60%, about 60% to about 70%, about 70% to about 80%, about 90% toabout 90%, or about 90% to about 99% (volume) nitrogen.

In some embodiments, the portion of the gas phase other than isoprenegas comprises between about 1% to about 50% (volume) CO₂, such asbetween about 1% to about 10%, about 10% to about 20%, about 20% toabout 30%, about 30% to about 40%, or about 40% to about 50% (volume)CO₂.

In some embodiments, an isoprene composition also contains ethanol. Forexample, ethanol may be used for extractive distillation of isoprene,resulting in compositions (such as intermediate product streams) thatinclude both ethanol and isoprene. Desirably, the amount of ethanol isoutside the flammability envelope for ethanol. The LOC of ethanol isabout 8.7 vol %, and the LFL for ethanol is about 3.3 vol % at standardconditions, such as about 1 atmosphere and about 6° F. (NFPA 69 Standardon Explosion Prevention Systems, 2008 edition, which is herebyincorporated by reference in its entirety, particularly with respect toLOC, LFL, and UFL values). In some embodiments, compositions thatinclude isoprene and ethanol are produced in the presence of less thanthe LOC required to have a flammable mixture of ethanol (such as lessthan about 8.7% vol %). In some embodiments in which compositions thatinclude isoprene and ethanol are produced in the presence of greaterthan or about the LOC required to have a flammable mixture of ethanol,the ethanol concentration is below the LFL (such as less than about 3.3vol. %).

In various embodiments, the amount of oxidant (such as oxygen) is belowthe LOC of any fuel in the system (such as isoprene or ethanol). Invarious embodiments, the amount of oxidant (such as oxygen) is less thanabout 60, 40, 30, 20, 10, or 5% of the LOC of isoprene or ethanol. Invarious embodiments, the amount of oxidant (such as oxygen) is less thanthe LOC of isoprene or ethanol by at least 2, 4, 5, or more absolutepercentage points (vol %). In particular embodiments, the amount ofoxygen is at least 2 absolute percentage points (vol %) less than theLOC of isoprene or ethanol (such as an oxygen concentration of less than7.5 vol % when the LOC of isoprene is 9.5 vol %). In variousembodiments, the amount of fuel (such as isoprene or ethanol) is lessthan or about 25, 20, 15, 10, or 5% of the LFL for that fuel.

High Efficiency Production and Recovery of Isoprene, a VolatileHydrocarbon, by Fermentation

Methods are provided herein of producing isoprene comprising a)culturing cells under suitable conditions for production of isoprene;and b) producing isoprene, wherein the liquid phase concentration ofisoprene is less than about 200 mg/L. In some embodiments, the liquidphase concentration of isoprene in the culture is less than about any of175 mg/L, 150 mg/L, 125 mg/L, 100 mg/L, 75 mg/L, 50 mg/L, 25 mg/L, 20mg/L, 15 mg/L, 10 mg/L, 5 mg/L, or 2.5 mg/L. In some embodiments, theliquid phase concentration of isoprene in culture is between about anyof 0.1 mg/L to 200 mg/L, 1 mg/L to 200 mg/L, 1 mg/L to 150 mg/L, 1 mg/Lto 100 mg/L, 1 mg/L to 50 mg/L, 1 mg/L to 25 mg/L, 1 mg/L to 20 mg/L, or10 mg/L to 20 mg/L. In some embodiments, the isoprene produced is anyconcentration or amount disclosed in the section entitled “ExemplaryProduction of Isoprene.” In some embodiments, the liquid phaseconcentration is below the solubility limit of isoprene.

In some embodiments of the methods, the cells produce greater than about400 nmole/g_(wcm)/hour of isoprene. In some embodiments, the amount ofisoprene is between about any of 400 nmole/g_(wcm)/hour to 1mole/g_(wcm)/hour, 400 nmole/g_(wcm)/hour to 1 mmole/g_(wcm)/hour, 400nmole/g_(wcm)/hour to 40 mmole/g_(wcm)/hour, 400 nmole/g_(wcm)/hour to 4mmole/g_(wcm)/hour, 1 mmole/g_(wcm)/hour to 1.5 mmole/g_(wcm)/hour, 1.5mmole/g_(wcm)/hour to 3 mmole/g_(wcm)/hour, 3 mmole/g_(wcm)/hour to 5mmole/g_(wcm)/hour, 5 mmole/g_(wcm)/hour to 25 mmole/g_(wcm)/hour, 25mmole/g_(wcm)/hour to 100 mmole/g_(wcm)/hour, 100 mmole/g_(wcm)/hour to500 mmole/g_(wcm)/hour, or 500 mmole/g_(wcm)/hour to 1000mmole/g_(wcm)/hour. In some embodiments, the amount of isoprene is aboutany of 1 mmole/g_(wcm)/hour, 1.5 mmole/g_(wcm)/hour, 2mmole/g_(wcm)/hour, 3 mmole/g_(wcm)/hour, 4 mmole/g_(wcm)/hour, or 5mmole/g_(wcm)/hour.

The low value for Henry's coefficient (in M atm⁻¹ units) means thatisoprene can be recovered from fermentation broth by gas stripping atlow sparging rates, for example 0.01 vvm to 2 vvm. In some embodiments,the gas sparging rate is between about any of 0.1 vvm to 1 vvm, 0.01 vvmto 0.5 vvm, 0.2 vvm to 1 vvm, or 0.5 vvm to 1 vvm. In some embodiments,the gas sparging rate is about any of 0.1 vvm, 0.25 vvm, 0.5 vvm, 0.75vvm, 1 vvm, 1.25 vvm, 1.5 vvm, 1.75 vvm, or 2 vvm. In some embodiments,the low sparging rates are maintained for the entire course of thefermentation run, during growth phase, or during stationary phase. Insome embodiments, the low sparging rates are maintained for betweenabout any of 1 hour to 5 hours, 5 hours to 10 hours, 10 hours to 20hours, 20 hours to 30 hours, 30 hours to 40 hours, 40 hours to 50 hours,or 50 hours to 60 hours. The lower desirable gas sparge limit is definedby the point at which the aqueous phase becomes saturated with isopreneand a liquid organic phase forms. This can only occur below the boilingpoint of isoprene (34.1° C. at 1 atm), above which a liquid isoprenephase will never form. At temperatures below the boiling point ofisoprene, the formation of a liquid phase is determined by the aqueoussolubility of isoprene, which is approximately 650 mg/L at 25° C. Whileit is highly desirable to avoid the formation of a liquid isoprenephase, it is not absolutely required provided that the cells cantolerate the presence of liquid isoprene without toxic effects.

In some embodiments, the oxygen, CO₂, and isoprene are any of theamounts or concentrations discussed in the section entitled “Productionof Isoprene with Safe Operating Ranges.” In some embodiments, all theoxygen is consumed by the cells while maintaining fully aerobicmetabolism. In some embodiments, an excess of oxygen is used in order tosatisfy the oxygen demands of the cells. Desirable ranges of oxygen inthe off-gas are less than 20%, or less than 15% or less than 10% (v/v).Levels of oxygen below the limiting oxygen concentration required forcombustion of isoprene (9.5% v/v at 1 atm) are particularly desirable.In some embodiments, oxygen-enriched air is utilized with the purpose ofallowing minimal gas sweep rates while satisfying the cellular oxygendemand. In some embodiments, the portion of the gas phase of the gassweep comprises between about 0.1% to about 10%, about 10% to about 20%,or about 20% to about 30% (volume) oxygen. In some embodiments, isoprenefermentations are performed under high pressure in order minimize theamount of excess oxygen required to maintain the required dissolvedoxygen levels in the liquid phase.

In some embodiments, the reduction of the gas sweep rate through thefermentor is advantageous for an integrated isoprene production processin that such conditions enrich the off-gas isoprene levels up to about30,000 μg/L (about 1% v/v) without adversely affecting the physiology ofthe cells.

In some embodiments, reduced gas-sparge rates do not significantlyadversely affect the physiology of the cells. In some embodiments, thecarbon dioxide evolution rate of cells in culture with reducedgas-sparge rates is between about any of 1×10⁻¹⁸ mmol/L/hour to about 1mol/L/hour, 1 mmol/L/hour to 1 mol/L/hour, 25 mmol/L/hour to 750mmol/L/hour, 25 mmol/L/hour to 75 mmol/L/hour, 250 mmol/L/hour to 750mmol/L/hour, or 450 mmol/L/hour to 550 mmol/L/hour. In some embodiments,the carbon dioxide evolution rate is about any of 50 mmol/L/hour, 100mmol/L/hour, 150 mmol/L/hour, 200 mmol/L/hour, 250 mmol/L/hour, 300mmol/L/hour, 350 mmol/L/hour, 400 mmol/L/hour, 450 mmol/L/hour, or 500mmol/L/hour. In some embodiments, cell viability with reduced gas-spargerates is reduced by less than about any of 1.75-fold, 1.5-fold,1.25-fold, 1-fold, 0.75-fold, 0.5-fold, or 0.25-fold. In someembodiments, cell viability with reduced gas-sparge rates is reduced byabout 2-fold. In some embodiments, cell viability with reducedgas-sparge rates of a cell expressing a MVA pathway and/or DXP pathwayRNA and/or protein from one or more of a heterologous and/or duplicatecopy of a MVA pathway and/or DXP pathway nucleic acid is compared to acontrol cell lacking one or more of a heterologous and/or duplicate copyof a MVA pathway and/or DXP pathway nucleic acid with reduced gas-spargerates. In some embodiments, cell viability with reduced gas-sparge ratesof a cell expressing a MVA pathway and/or DXP pathway RNA and/or proteinfrom one or more of a heterologous and/or duplicate copy of a MVApathway and/or DXP pathway nucleic acid under the control of aninducible promoter, wherein the promotor is induced, is compared to acontrol cell containing one or more of a heterologous and/or duplicatecopy of a MVA pathway and/or DXP pathway nucleic acid under the controlof an inducible promoter, wherein the promotor is not induced(uninduced) with reduced gas-sparge rates. In some embodiments, theinducible promoter is a beta-galactosidase promotor.

In some embodiments, the fermentation of a genetically modified hostorganism that converts at least 5% of the total carbon consumed by theorganism into a volatile, unsaturated hydrocarbon. In some embodiments,the production of an unsaturated hydrocarbon at such a rate as to bepresent in the fermentation off-gas at a level of at least about any of100 μg/L, 500 μg/L, 1000 μg/L, 2, 500 μg/L, 5,000 μg/L, 7,500 μg/L, or10,000 μg/L.

In some embodiments, the unsaturated hydrocarbon is recovered from theoff-gas stream in a manner that is suited to high-rates of production,which correspond to concentrations in the off-gas of at least about anyof 100 μg/L, 500 μg/L, 1000 μg/L, 2,500 μg/L, 5,000 μg/L, 7,500 μg/L, or10,000 μg/L. In some embodiments, the continuous extraction and recoveryof an unsaturated hydrocarbon from the fermentation off-gas inparticular at low gas sweep rates such that the resulting off-gas isenriched in the volatile component of interest. In some embodiments,recovery of the volatile hydrocarbon by methods that depend on elevatedconcentrations of the volatile. For example, efficient capture ofisoprene in fermentation off-gas through the use ofcompression/condensation or extractive distillation technologies. Alsocontemplated is the use of activated carbon cartridges in addition tosilica gel adsorbants, desorption and concentration of isoprene fromcarbon cartridges, and/or construction and fermentation of hostorganisms such as E. coli strains that can convert about 5% or more ofthe glucose substrate to isoprene and result in off-gas concentrationsof greater than about 15,000 μg/L isoprene. Recovery methods include anyof the methods described herein.

Also provided herein are methods of producing a compound, wherein thecompound has one or more characteristics selected from the groupconsisting of (a) a Henry's law coefficient of less than about 250 M/atmand (b) a solubility in water of less than about 100 g/L. In someembodiments, the method comprises: a) culturing cells under suitableconditions for production of the compound, wherein gas is added (such asthe addition of gas to a system such as a fermentation system) at a gassparging rate between about 0.01 vvm to about 2 vvm; and b) producingthe compound.

In some embodiments, the amount of the compound that partitions into thecell mass is not included in the liquid phase solubility values. In someembodiments, the liquid phase concentration is below the solubilitylimit of compound.

In some embodiments, the compounds can be continuously recovered fromfermentation broth by gas stripping at moderate to low gas spargingrates, in particular those compounds with Henry's law coefficients ofabout any of less than 250 M/atm, 200 M/atm, 150 M/atm, 100 M/atm, 75M/atm, 50 M/atm, 25 M/atm, 10 M/atm, 5 M/atm, or 1 M/atm. Examplesinclude aldehydes such as acetaldehyde (15 M/atm), ketones such asacetone (30 M/atm) or 2-butanone (20 M/atm), or alcohols includingmethanol (220 M/atm), ethanol (200 M/atm), 1-butanol (120 M/atm) or C5alcohols including 3-methyl-3-buten-1-ol, and 3-methyl-2-buten-1-ol(50-100 M/atm). Esters of alcohols generally have lower Henry'sconstants than the respective alcohols, for example ethyl acetate (6-9M/atm) or the acetyl esters of C5 alcohols (<5 M/atm). Compounds withHenry's law coefficients of less than 1 M/atm are particularlydesirable. Examples include hemiterpenes, monoterpenes, orsesquiterpenes, in addition to other hydrocarbons such as C1 to C5hydrocarbons (e.g., methane, ethane, ethylene, or propylene). In someembodiments, the hydrocarbons such as C1 to C5 hydrocarbons aresaturated, unsaturated, or branched.

In general, there is a correlation between Henry's law coefficient andwater solubility in that compounds with very low coefficients aresparingly soluble in water (substantially water insoluble). Althoughvolatiles with infinite solubilities in water (e.g. acetone or ethanol)can be removed by gas stripping, desirable solubility limits are lessthan about any of 100 g/L, 75 g/L, 50 g/L, 25 g/L, 10 g/L, 5 g/L, or 1g/L.

In some embodiments of any of the methods of producing any of thecompounds described above, the gas sparging rate is between about any of0.1 vvm to 1 vvm, 0.2 vvm to 1 vvm, or 0.5 vvm to 1 vvm. In someembodiments, the gas sparging rate is about any of 0.1 vvm, 0.25 vvm,0.5 vvm, 0.75 vvm, 1 vvm, 1.25 vvm, 1.5 vvm, 1.75 vvm, or 2 vvm. In someembodiments, the low sparging rates are maintained for the entire courseof the fermentation run, during growth phase, or during stationaryphase. In some embodiments, the low sparging rates are maintained forbetween about any of 1 hour to 5 hours, 5 hours to 10 hours, 10 hours to20 hours, 20 hours to 30 hours, 30 hours to 40 hours, 40 hours to 50hours, or 50 hours to 60 hours.

Any of the systems described herein can be used in the methods ofproducing a compound described above. Standard methods would be used topurify such as those described in the section entitled “ExemplaryPurification Methods.” Separation can be performed post-recovery forexample, by distillation or selective adsorption techniques.

Exemplary Production of BioIsoprene

In some embodiments, the cells are cultured in a culture medium underconditions permitting the production of isoprene by the cells.

By “peak absolute productivity” is meant the maximum absolute amount ofisoprene in the off-gas during the culturing of cells for a particularperiod of time (e.g., the culturing of cells during a particularfermentation run). By “peak absolute productivity time point” is meantthe time point during a fermentation run when the absolute amount ofisoprene in the off-gas is at a maximum during the culturing of cellsfor a particular period of time (e.g., the culturing of cells during aparticular fermentation run). In some embodiments, the isoprene amountis measured at the peak absolute productivity time point. In someembodiments, the peak absolute productivity for the cells is about anyof the isoprene amounts disclosed herein.

By “peak specific productivity” is meant the maximum amount of isopreneproduced per cell during the culturing of cells for a particular periodof time (e.g., the culturing of cells during a particular fermentationrun). By “peak specific productivity time point” is meant the time pointduring the culturing of cells for a particular period of time (e.g., theculturing of cells during a particular fermentation run) when the amountof isoprene produced per cell is at a maximum. The peak specificproductivity is determined by dividing the total productivity by theamount of cells, as determined by optical density at 600 nm (OD₆₀₀). Insome embodiments, the isoprene amount is measured at the peak specificproductivity time point. In some embodiments, the peak specificproductivity for the cells is about any of the isoprene amounts per celldisclosed herein.

By “peak volumetric productivity” is meant the maximum amount ofisoprene produced per volume of broth (including the volume of the cellsand the cell medium) during the culturing of cells for a particularperiod of time (e.g., the culturing of cells during a particularfermentation run). By “peak specific volumetric productivity time point”is meant the time point during the culturing of cells for a particularperiod of time (e.g., the culturing of cells during a particularfermentation run) when the amount of isoprene produced per volume ofbroth is at a maximum. The peak specific volumetric productivity isdetermined by dividing the total productivity by the volume of broth andamount of time. In some embodiments, the isoprene amount is measured atthe peak specific volumetric productivity time point. In someembodiments, the peak specific volumetric productivity for the cells isabout any of the isoprene amounts per volume per time disclosed herein.

By “peak concentration” is meant the maximum amount of isoprene producedduring the culturing of cells for a particular period of time (e.g., theculturing of cells during a particular fermentation run). By “peakconcentration time point” is meant the time point during the culturingof cells for a particular period of time (e.g., the culturing of cellsduring a particular fermentation run) when the amount of isopreneproduced per cell is at a maximum. In some embodiments, the isopreneamount is measured at the peak concentration time point. In someembodiments, the peak concentration for the cells is about any of theisoprene amounts disclosed herein.

By “average volumetric productivity” is meant the average amount ofisoprene produced per volume of broth (including the volume of the cellsand the cell medium) during the culturing of cells for a particularperiod of time (e.g., the culturing of cells during a particularfermentation run). The average volumetric productivity is determined bydividing the total productivity by the volume of broth and amount oftime. In some embodiments, the average specific volumetric productivityfor the cells is about any of the isoprene amounts per volume per timedisclosed herein.

By “cumulative total productivity” is meant the cumulative, total amountof isoprene produced during the culturing of cells for a particularperiod of time (e.g., the culturing of cells during a particularfermentation run). In some embodiments, the cumulative, total amount ofisoprene is measured. In some embodiments, the cumulative totalproductivity for the cells is about any of the isoprene amountsdisclosed herein.

As used herein, “relative detector response” refers to the ratio betweenthe detector response (such as the GC/MS area) for one compound (such asisoprene) to the detector response (such as the GC/MS area) of one ormore compounds (such as all C5 hydrocarbons). The detector response maybe measured as described herein, such as the GC/MS analysis performedwith an Agilent 6890 GC/MS system fitted with an Agilent HP-5MS GC/MScolumn (30 m×250 μm; 0.25 μm film thickness). If desired, the relativedetector response can be converted to a weight percentage using theresponse factors for each of the compounds. This response factor is ameasure of how much signal is generated for a given amount of aparticular compound (that is, how sensitive the detector is to aparticular compound). This response factor can be used as a correctionfactor to convert the relative detector response to a weight percentagewhen the detector has different sensitivities to the compounds beingcompared. Alternatively, the weight percentage can be approximated byassuming that the response factors are the same for the compounds beingcompared. Thus, the weight percentage can be assumed to be approximatelythe same as the relative detector response.

In some embodiments, the cells in culture produce isoprene at greaterthan or about 1, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600,700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000,5,000, 10,000, 12,500, 20,000, 30,000, 40,000, 50,000, 75,000, 100,000,125,000, 150,000, 188,000, or more nmole of isoprene/gram of cells forthe wet weight of the cells/hour (nmole/g_(wcm)/hr). In someembodiments, the amount of isoprene is between about 2 to about 200,000nmole/g_(wcm)/hr, such as between about 2 to about 100 nmole/g_(wcm)/hr,about 100 to about 500 nmole/g_(wcm)/hr, about 150 to about 500nmole/g_(wcm)/hr, about 500 to about 1,000 nmole/g_(wcm)/hr, about 1,000to about 2,000 nmole/g_(wcm)/hr, or about 2,000 to about 5,000nmole/g_(wcm)/hr, about 5,000 to about 10,000 nmole/g_(wcm)/hr, about10,000 to about 50,000 nmole/g_(wcm)/hr, about 50,000 to about 100,000nmole/g_(wcm)/hr, about 100,000 to about 150,000 nmole/g_(wcm)/hr, orabout 150,000 to about 200,000 nmole/g_(wcm)/hr. In some embodiments,the amount of isoprene is between about 20 to about 5,000nmole/g_(wcm)/hr, about 100 to about 5,000 nmole/g_(wcm)/hr, about 200to about 2,000 nmole/g_(wcm)/hr, about 200 to about 1,000nmole/g_(wcm)/hr, about 300 to about 1,000 nmole/g_(wcm)/hr, or about400 to about 1,000 nmole/g_(wcm)/hr, about 1,000 to about 5,000nmole/g_(wcm)/hr, about 2,000 to about 20,000 nmole/g_(wcm)/hr, about5,000 to about 50,000 nmole/g_(wcm)/hr, about 10,000 to about 100,000nmole/g_(wcm)/hr, about 20,000 to about 150,000 nmole/g_(wcm)/hr, orabout 20,000 to about 200,000 nmole/g_(wcm)/hr.

The amount of isoprene in units of nmole/g_(wcm)/hr can be measured asdisclosed in U.S. Pat. No. 5,849,970, which is hereby incorporated byreference in its entirety, particularly with respect to the measurementof isoprene production. For example, two mL of headspace (e.g.,headspace from a culture such as 2 mL of culture cultured in sealedvials at 32° C. with shaking at 200 rpm for approximately 3 hours) areanalyzed for isoprene using a standard gas chromatography system, suchas a system operated isothermally (85° C.) with an n-octane/porasil Ccolumn (Alltech Associates, Inc., Deerfield, Ill.) and coupled to a RGD2mercuric oxide reduction gas detector (Trace Analytical, Menlo Park,Calif.) (see, for example, Greenberg et al, Atmos. Environ. 27A:2689-2692, 1993; Silver et al., Plant Physiol. 97:1588-1591, 1991, whichare each hereby incorporated by reference in their entireties,particularly with respect to the measurement of isoprene production).The gas chromatography area units are converted to nmol isoprene via astandard isoprene concentration calibration curve. In some embodiments,the value for the grams of cells for the wet weight of the cells iscalculated by obtaining the A₆₀₀ value for a sample of the cell culture,and then converting the A₆₀₀ value to grams of cells based on acalibration curve of wet weights for cell cultures with a known A₆₀₀value. In some embodiments, the grams of the cells is estimated byassuming that one liter of broth (including cell medium and cells) withan A₆₀₀ value of 1 has a wet cell weight of 1 gram. The value is alsodivided by the number of hours the culture has been incubating for, suchas three hours.

In some embodiments, the cells in culture produce isoprene at greaterthan or about 1, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600,700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000,5,000, 10,000, 100,000, or more ng of isoprene/gram of cells for the wetweight of the cells/hr (ng/g_(wcm)/h). In some embodiments, the amountof isoprene is between about 2 to about 5,000 ng/g_(wcm)/h, such asbetween about 2 to about 100 ng/g_(wcm)/h, about 100 to about 500ng/g_(wcm)/h, about 500 to about 1,000 ng/g_(wcm)/h, about 1,000 toabout 2,000 ng/g_(wcm)/h, or about 2,000 to about 5,000 ng/g_(wcm)/h. Insome embodiments, the amount of isoprene is between about 20 to about5,000 ng/g_(wcm)/h, about 100 to about 5,000 ng/g_(wcm)/h, about 200 toabout 2,000 ng/g_(wcm)/h, about 200 to about 1,000 ng/g_(wcm)/h, about300 to about 1,000 ng/g_(wcm)/h, or about 400 to about 1,000ng/g_(wcm)/h. The amount of isoprene in ng/g_(wcm)/h can be calculatedby multiplying the value for isoprene production in the units ofnmole/g_(wcm)/hr discussed above by 68.1 (as described in Equation 5below).

In some embodiments, the cells in culture produce a cumulative titer(total amount) of isoprene at greater than or about 1, 10, 25, 50, 100,150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500,1,750, 2,000, 2,500, 3,000, 4,000, 5,000, 10,000, 50,000, 100,000, ormore mg of isoprene/L of broth (mg/L_(broth), wherein the volume ofbroth includes the volume of the cells and the cell medium). In someembodiments, the amount of isoprene is between about 2 to about 5,000mg/L_(broth), such as between about 2 to about 100 mg/L_(broth), about100 to about 500 mg/L_(broth), about 500 to about 1,000 mg/L_(broth),about 1,000 to about 2,000 mg/L_(broth), or about 2,000 to about 5,000mg/L_(broth). In some embodiments, the amount of isoprene is betweenabout 20 to about 5,000 mg/L_(broth), about 100 to about 5,000mg/L_(broth), about 200 to about 2,000 mg/L_(broth), about 200 to about1,000 mg/L_(broth), about 300 to about 1,000 mg/L_(broth), or about 400to about 1,000 mg/L_(broth).

The specific productivity of isoprene in mg of isoprene/L of headspacefrom shake flask or similar cultures can be measured by taking a 1 mlsample from the cell culture at an OD₆₀₀ value of approximately 1.0,putting it in a 20 mL vial, incubating for 30 minutes, and thenmeasuring the amount of isoprene in the headspace (as described, forexample, in Example I, part II). If the OD₆₀₀ value is not 1.0, then themeasurement can be normalized to an OD₆₀₀ value of 1.0 by dividing bythe OD₆₀₀ value. The value of mg isoprene/L headspace can be convertedto mg/L_(broth)/hr/OD₆₀₀ of culture broth by multiplying by a factor of38. The value in units of mg/L_(broth)/hr/OD₆₀₀ can be multiplied by thenumber of hours and the OD₆₀₀ value to obtain the cumulative titer inunits of mg of isoprene/L of broth.

In some embodiments, the cells in culture have an average volumetricproductivity of isoprene at greater than or about 0.1, 1.0, 10, 25, 50,100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 1100,1200, 1300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,100,2,200, 2,300, 2,400, 2,500, 2,600, 2,700, 2,800, 2,900, 3,000, 3,100,3,200, 3,300, 3,400, 3,500, or more mg of isoprene/L of broth/hr(mg/L_(broth)/hr, wherein the volume of broth includes the volume of thecells and the cell medium). In some embodiments, the average volumetricproductivity of isoprene is between about 0.1 to about 3,500mg/L_(broth)/hr, such as between about 0.1 to about 100 mg/L_(broth)/hr,about 100 to about 500 mg/L_(broth)/hr, about 500 to about 1,000mg/L_(broth)/hr, about 1,000 to about 1,500 mg/L_(broth)/hr, about 1,500to about 2,000 mg/L_(broth)/hr, about 2,000 to about 2,500mg/L_(broth)/hr, about 2,500 to about 3,000 mg/L_(broth)/hr, or about3,000 to about 3,500 mg/L_(broth)/hr. In some embodiments, the averagevolumetric productivity of isoprene is between about 10 to about 3,500mg/L_(broth)/hr, about 100 to about 3,500 mg/L_(broth)/hr, about 200 toabout 1,000 mg/L_(broth)/hr, about 200 to about 1,500 mg/L_(broth)/hr,about 1,000 to about 3,000 mg/L_(broth)/hr, or about 1,500 to about3,000 mg/L_(broth)/hr.

In some embodiments, the cells in culture have a peak volumetricproductivity of isoprene at greater than or about 0.5, 1.0, 10, 25, 50,100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 1100,1200, 1300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,100,2,200, 2,300, 2,400, 2,500, 2,600, 2,700, 2,800, 2,900, 3,000, 3,100,3,200, 3,300, 3,400, 3,500, 3,750, 4,000, 4,250, 4,500, 4,750, 5,000,5,250, 5,500, 5,750, 6,000, 6,250, 6,500, 6,750, 7,000, 7,250, 7,500,7,750, 8,000, 8,250, 8,500, 8,750, 9,000, 9,250, 9,500, 9,750, 10,000,12,500, 15,000, or more mg of isoprene/L of broth/hr (mg/L_(broth)/hr,wherein the volume of broth includes the volume of the cells and thecell medium). In some embodiments, the peak volumetric productivity ofisoprene is between about 0.5 to about 15,000 mg/L_(broth)/hr, such asbetween about 0.5 to about 10 mg/L_(broth)/hr, about 1.0 to about 100mg/L_(broth)/hr, about 100 to about 500 mg/L_(broth)/hr, about 500 toabout 1,000 mg/L_(broth)/hr, about 1,000 to about 1,500 mg/L_(broth)/hr,about 1,500 to about 2,000 mg/L_(broth)/hr, about 2,000 to about 2,500mg/L_(broth)/hr, about 2,500 to about 3,000 mg/L_(broth)/hr, about 3,000to about 3,500 mg/L_(broth)/hr, about 3,500 to about 5,000mg/L_(broth)/hr, about 5,000 to about 7,500 mg/L_(broth)/hr, about 7,500to about 10,000 mg/L_(broth)/hr, about 10,000 to about 12,500mg/L_(broth)/h, or about 12,500 to about 15,000 mg/L_(broth)/hr. In someembodiments, the peak volumetric productivity of isoprene is betweenabout 10 to about 15,000 mg/L_(broth)/hr, about 100 to about 2,500mg/L_(broth)/hr, about 1,000 to about 5,000 mg/L_(broth)/hr, about 2,500to about 7,500 mg/L_(broth)/hr, about 5,000 to about 10,000mg/L_(broth)/hr, about 7,500 to about 12,500 mg/L_(broth)/hr, or about10,000 to about 15,000 mg/L_(broth)/hr.

The instantaneous isoprene production rate in mg/L_(broth)/hr in afermentor can be measured by taking a sample of the fermentor off-gas,analyzing it for the amount of isoprene (in units such as mg of isopreneper L_(gas)) as described, for example, in Example I, part II andmultiplying this value by the rate at which off-gas is passed thougheach liter of broth (e.g., at 1 vvm (volume of air/volume ofbroth/minute) this is 60 L_(gas) per hour). Thus, an off-gas level of 1mg/L_(gas) corresponds to an instantaneous production rate of 60mg/L_(broth)/hr at air flow of 1 vvm. If desired, the value in the unitsmg/L_(broth)/hr can be divided by the OD₆₀₀ value to obtain the specificrate in units of mg/L_(broth)/hr/OD. The average value of mgisoprene/L_(gas) can be converted to the total product productivity(grams of isoprene per liter of fermentation broth, mg/L_(broth)) bymultiplying this average off-gas isoprene concentration by the totalamount of off-gas sparged per liter of fermentation broth during thefermentation. Thus, an average off-gas isoprene concentration of 0.5mg/L_(broth)/hr over 10 hours at 1 vvm corresponds to a total productconcentration of 300 mg isoprene/L_(broth).

In some embodiments, the cells in culture convert greater than or about0.0015, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.12, 0.14, 0.16, 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4,2.6, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, 14.0,15.0, 16.0, 17.0, 18.0, 19.0, 20.0, 21.0, 22.0, 23.0, 23.2, 23.4, 23.6,23.8, 24.0, 25.0, 30.0, 31.0, 32.0, 33.0, 35.0, 37.5, 40.0, 45.0, 47.5,50.0, 55.0, 60.0, 65.0, 70.0, 75.0, 80.0, 85.0, or 90.0 molar % of thecarbon in the cell culture medium into isoprene. In some embodiments,the percent conversion of carbon into isoprene is between about 0.002 toabout 90.0 molar %, such as about 0.002 to about 0.005%, about 0.005 toabout 0.01%, about 0.01 to about 0.05%, about 0.05 to about 0.15%, 0.15to about 0.2%, about 0.2 to about 0.3%, about 0.3 to about 0.5%, about0.5 to about 0.8%, about 0.8 to about 1.0%, about 1.0 to about 1.6%,about 1.6 to about 3.0%, about 3.0 to about 5.0%, about 5.0 to about8.0%, about 8.0 to about 10.0%, about 10.0 to about 15.0%, about 15.0 toabout 20.0%, about 20.0 to about 25.0%, about 25.0% to 30.0%, about30.0% to 35.0%, about 35.0% to 40.0%, about 45.0% to 50.0%, about 50.0%to 55.0%, about 55.0% to 60.0%, about 60.0% to 65.0%, about 65.0% to70.0%, about 75.0% to 80.0%, about 80.0% to 85.0%, or about 85.0% to90.0%. In some embodiments, the percent conversion of carbon intoisoprene is between about 0.002 to about 0.4 molar %, 0.002 to about0.16 molar %, 0.04 to about 0.16 molar %, about 0.005 to about 0.3 molar%, about 0.01 to about 0.3 molar %, about 0.05 to about 0.3 molar %,about 0.1 to 0.3 molar %, about 0.3 to about 1.0 molar %, about 1.0 toabout 5.0 molar %, about 2 to about 5.0 molar %, about 5.0 to about 10.0molar %, about 7 to about 10.0 molar %, about 10.0 to about 20.0 molar%, about 12 to about 20.0 molar %, about 16 to about 20.0 molar %, about18 to about 20.0 molar %, about 18 to 23.2 molar %, about 18 to 23.6molar %, about 18 to about 23.8 molar %, about 18 to about 24.0 molar %,about 18 to about 25.0 molar %, about 20 to about 30.0 molar %, about 30to about 40.0 molar %, about 30 to about 50.0 molar %, about 30 to about60.0 molar %, about 30 to about 70.0 molar %, about 30 to about 80.0molar %, or about 30 to about 90.0 molar %.

The percent conversion of carbon into isoprene (also referred to as “%carbon yield”) can be measured by dividing the moles carbon in theisoprene produced by the moles carbon in the carbon source (such as themoles of carbon in batched and fed glucose and yeast extract). Thisnumber is multiplied by 100% to give a percentage value (as indicated inEquation 1).

% Carbon Yield=(moles carbon in isoprene produced)/(moles carbon incarbon source)*100  Equation 1

For this calculation, yeast extract can be assumed to contain 50% w/wcarbon. As an example, for the 500 liter described in Example 7, partVIII, the percent conversion of carbon into isoprene can be calculatedas shown in Equation 2.

% Carbon Yield=(39.1 g isoprene*1/68.1 mol/g*5 C/mol)/[(181221 gglucose*1/180 mol/g*6 C/mol)+(17780 g yeast extract*0.5*1/12mol/g)]*100=0.042%  Equation 2

For the two 500 liter fermentations described herein (Example 7, partsVII and VIII), the percent conversion of carbon into isoprene wasbetween 0.04-0.06%. A 0.11-0.16% carbon yield has been achieved using 14liter systems as described herein. Example 11, part V describes the1.53% conversion of carbon to isoprene using the methods describedherein.

One skilled in the art can readily convert the rates of isopreneproduction or amount of isoprene produced into any other units.Exemplary equations are listed below for interconverting between units.

Units for Rate of Isoprene Production (Total and Specific)

1 g isoprene/L_(broth)/hr=14.7 mmol isoprene/L_(broth)/hr(totalvolumetric rate)  Equation 3

1 nmol isoprene/g_(wcm)/hr=1 nmol isoprene/L_(broth)/hr/OD₆₀₀ (Thisconversion assumes that one liter of broth with an OD₆₀₀ value of 1 hasa wet cell weight of 1 gram.)  Equation 4

1 nmol isoprene/g_(wcm)/hr=68.1 ng isoprene/g_(wcm)/hr (given themolecular weight of isoprene)  Equation 5

1 nmol isoprene/L_(gas) O₂/hr=90 nmol isoprene/L_(broth)/hr (at an O₂flow rate of 90 L/hr per L of culture broth)  Equation 6

1 μg isoprene/L_(gas) isoprene in off-gas=60 μg isoprene/L_(broth)/hr ata flow rate of 60 L_(gas) per L_(broth)(1 vvm)  Equation 7

Units for Titer (Total and Specific)

1 nmol isoprene/mg cell protein=150 nmol isoprene/L_(broth)/OD₆₀₀ (Thisconversion assumes that one liter of broth with an OD₆₀₀ value of 1 hasa total cell protein of approximately 150 mg) (specificproductivity)  Equation 8

1 g isoprene/L_(broth)=14.7 mmol isoprene/L_(broth)(totaltiter)  Equation 9

If desired, Equation 10 can be used to convert any of the units thatinclude the wet weight of the cells into the corresponding units thatinclude the dry weight of the cells.

Dry weight of cells=(wet weight of cells)/3.3  Equation 10

If desired, Equation 11 can be used to convert between units of ppm andμg/L. In particular, “ppm” means parts per million defined in terms ofμg/g (w/w). Concentrations of gases can also be expressed on avolumetric basis using “ppmv” (parts per million by volume), defined interms of μL/L (vol/vol). Conversion of μg/L to ppm (e.g., μg of analyteper g of gas) can be performed by determining the mass per L of off-gas(i.e., the density of the gas). For example, a liter of air at standardtemperature and pressure (STP; 101.3 kPa (1 bar) and 273.15K). has adensity of approximately 1.29 g/L. Thus, a concentration of 1 ppm (μg/g)equals 1.29 μg/L at STP (equation 11). The conversion of ppm (μg/g) toμg/L is a function of both pressure, temperature, and overallcomposition of the off-gas.

1 ppm (μg/g) equals 1.29 μg/L at standard temperature and pressure (STP;101.3 kPa (1 bar) and 273.15K).  Equation 11

Conversion of μg/L to ppmv (e.g., μL of analyte per L of gas) can beperformed using the Universal Gas Law (equation 12). For example, anoff-gas concentration of 1000 μg/L_(gas) corresponds to 14.7μmol/L_(gas). The universal gas constant is 0.082057 L.atm K⁻¹mol⁻¹, sousing equation 12, the volume occupied by 14.7 μmol of HG at STP isequal to 0.329 mL. Therefore, the concentration of 1000 μg/L HG is equalto 329 ppmv or 0.0329% (v/v) at STP.

PV=nRT, where “P” is pressure, “V” is volume, “n” is moles of gas, “R”is the Universal gas constant, and “T” is temperature inKelvin.  Equation 12

The amount of impurities in isoprene compositions are typically measuredherein on a weight per volume (w/v) basis in units such as μg/L. Ifdesired, measurements in units of μg/L can be converted to units ofmg/m³ using equation 13.

1 μg/L=1 mg/m³  Equation 13

In some embodiments described herein, a cell comprising a heterologousnucleic acid encoding an isoprene synthase polypeptide produces anamount of isoprene that is at least or about 2-fold, 3-fold, 5-fold,10-fold, 25-fold, 50-fold, 100-fold, 150-fold, 200-fold, 400-fold, orgreater than the amount of isoprene produced from a corresponding cellgrown under essentially the same conditions without the heterologousnucleic acid encoding the isoprene synthase polypeptide.

In some embodiments described herein, a cell comprising a heterologousnucleic acid encoding an isoprene synthase polypeptide and one or moreheterologous nucleic acids encoding a DXS, IDI, and/or MVA pathwaypolypeptide produces an amount of isoprene that is at least or about2-fold, 3-fold, 5-fold, 10-fold, 25-fold, 50-fold, 100-fold, 150-fold,200-fold, 400-fold, or greater than the amount of isoprene produced froma corresponding cell grown under essentially the same conditions withoutthe heterologous nucleic acids.

In some embodiments, the isoprene composition comprises greater than orabout 99.90, 99.92, 99.94, 99.96, 99.98, or 100% isoprene by weightcompared to the total weight of all C5 hydrocarbons in the composition.In some embodiments, the composition has a relative detector response ofgreater than or about 99.90, 99.91, 99.92, 99.93, 99.94, 99.95, 99.96,99.97, 99.98, 99.99, or 100% for isoprene compared to the detectorresponse for all C5 hydrocarbons in the composition. In someembodiments, the isoprene composition comprises between about 99.90 toabout 99.92, about 99.92 to about 99.94, about 99.94 to about 99.96,about 99.96 to about 99.98, about 99.98 to 100% isoprene by weightcompared to the total weight of all C5 hydrocarbons in the composition.

In some embodiments, the isoprene composition comprises less than orabout 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005,0.0001, 0.00005, or 0.00001% C5 hydrocarbons other than isoprene (suchas 1,3-cyclopentadiene, cis-1,3-pentadiene, trans-1,3-pentadiene,1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene, 2-methyl-1-butene,3-methyl-1-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, orcis-pent-3-ene-1-yne) by weight compared to the total weight of all C5hydrocarbons in the composition. In some embodiments, the compositionhas a relative detector response of less than or about 0.12, 0.10, 0.08,0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or0.00001% for C5 hydrocarbons other than isoprene compared to thedetector response for all C5 hydrocarbons in the composition. In someembodiments, the composition has a relative detector response of lessthan or about 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001,0.0005, 0.0001, 0.00005, or 0.00001% for 1,3-cyclopentadiene,cis-1,3-pentadiene, trans-1,3-pentadiene, 1,4-pentadiene, 1-pentyne,2-pentyne, 1-pentene, 2-methyl-1-butene, 3-methyl-1-butyne,pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-ynecompared to the detector response for all C5 hydrocarbons in thecomposition. In some embodiments, the isoprene composition comprisesbetween about 0.02 to about 0.04%, about 0.04 to about 0.06%, about 0.06to 0.08%, about 0.08 to 0.10%, or about 0.10 to about 0.12% C5hydrocarbons other than isoprene (such as 1,3-cyclopentadiene,cis-1,3-pentadiene, trans-1,3-pentadiene, 1,4-pentadiene, 1-pentyne,2-pentyne, 1-pentene, 2-methyl-1-butene, 3-methyl-1-butyne,pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne) byweight compared to the total weight of all C5 hydrocarbons in thecomposition.

In some embodiments, the isoprene composition comprises less than orabout 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 μg/L of acompound that inhibits the polymerization of isoprene for any compoundin the composition that inhibits the polymerization of isoprene. In someembodiments, the isoprene composition comprises between about 0.005 toabout 50, such as about 0.01 to about 10, about 0.01 to about 5, about0.01 to about 1, about 0.01 to about 0.5, or about 0.01 to about 0.005μg/L of a compound that inhibits the polymerization of isoprene for anycompound in the composition that inhibits the polymerization ofisoprene. In some embodiments, the isoprene composition comprises lessthan or about 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005μg/L of a hydrocarbon other than isoprene (such as 1,3-cyclopentadiene,cis-1,3-pentadiene, trans-1,3-pentadiene, 1,4-pentadiene, 1-pentyne,2-pentyne, 1-pentene, 2-methyl-1-butene, 3-methyl-1-butyne,pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne). Insome embodiments, the isoprene composition comprises between about 0.005to about 50, such as about 0.01 to about 10, about 0.01 to about 5,about 0.01 to about 1, about 0.01 to about 0.5, or about 0.01 to about0.005 lag/L of a hydrocarbon other than isoprene. In some embodiments,the isoprene composition comprises less than or about 50, 40, 30, 20,10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 μg/L of a protein or fatty acid(such as a protein or fatty acid that is naturally associated withnatural rubber).

In some embodiments, the isoprene composition comprises less than orabout 10, 5, 1, 0.8, 0.5, 0.1, 0.05, 0.01, or 0.005 ppm of alphaacetylenes, piperylenes, acetonitrile, or 1,3-cyclopentadiene. In someembodiments, the isoprene composition comprises less than or about 5, 1,0.5, 0.1, 0.05, 0.01, or 0.005 ppm of sulfur or allenes. In someembodiments, the isoprene composition comprises less than or about 30,20, 15, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 ppm of all acetylenes(such as 1-pentyne, 2-pentyne, 3-methyl-1-butyne, pent-4-ene-1-yne,trans-pent-3-ene-1-yne, and cis-pent-3-ene-1-yne). In some embodiments,the isoprene composition comprises less than or about 2000, 1000, 500,200, 100, 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 ppmof isoprene dimers, such as cyclic isoprene dimmers (e.g., cyclic C10compounds derived from the dimerization of two isoprene units).

In some embodiments, the isoprene composition includes ethanol, acetone,a C5 prenyl alcohol (such as 3-methyl-3-buten-1-ol or3-methyl-2-buten-1-ol), or any two or more of the foregoing. Inparticular embodiments, the isoprene composition comprises greater thanor about 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 20, 30, 40, 60, 80, 100,or 120 μg/L of ethanol, acetone, a C5 prenyl alcohol (such as3-methyl-3-buten-1-ol or 3-methyl-2-buten-1-ol), or any two or more ofthe foregoing. In some embodiments, the isoprene composition comprisesbetween about 0.005 to about 120, such as about 0.01 to about 80, about0.01 to about 60, about 0.01 to about 40, about 0.01 to about 30, about0.01 to about 20, about 0.01 to about 10, about 0.1 to about 80, about0.1 to about 60, about 0.1 to about 40, about 5 to about 80, about 5 toabout 60, or about 5 to about 40 μg/L of ethanol, acetone, a C5 prenylalcohol, or any two or more of the foregoing.

In some embodiments, the isoprene composition includes one or more ofthe following components: 2-heptanone, 6-methyl-5-hepten-2-one,2,4,5-trimethylpyridine, 2,3,5-trimethylpyrazine, citronellal,acetaldehyde, methanethiol, methyl acetate, 1-propanol, diacetyl,2-butanone, 2-methyl-3-buten-2-ol, ethyl acetate, 2-methyl-1-propanol,3-methyl-1-butanal, 3-methyl-2-butanone, 1-butanol, 2-pentanone,3-methyl-1-butanol, ethyl isobutyrate, 3-methyl-2-butenal, butylacetate, 3-methylbutyl acetate, 3-methyl-3-buten-1-yl acetate,3-methyl-2-buten-1-yl acetate, 3-hexen-1-ol, 3-hexen-1-yl acetate,limonene, geraniol(trans-3,7-dimethyl-2,6-octadien-1-ol), citronellol(3,7-dimethyl-6-octen-1-ol), (E)-3,7-dimethyl-1,3,6-octatriene,(Z)-3,7-dimethyl-1,3,6-octatriene, 2,3-cycloheptenolpyridine, or alinear isoprene polymer (such as a linear isoprene dimer or a linearisoprene trimer derived from the polymerization of multiple isopreneunits). In various embodiments, the amount of one of these componentsrelative to amount of isoprene in units of percentage by weight (i.e.,weight of the component divided by the weight of isoprene times 100) isgreater than or about 0.01, 0.02, 0.05, 0.1, 0.5, 1, 5, 10, 20, 30, 40,50, 60, 70, 80, 90, 100, or 110% (w/w). In some embodiments, therelative detector response for the second compound compared to thedetector response for isoprene is greater than or about 0.01, 0.02,0.05, 0.1, 0.5, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 110%.In various embodiments, the amount of one of these components relativeto amount of isoprene in units of percentage by weight (i.e., weight ofthe component divided by the weight of isoprene times 100) is betweenabout 0.01 to about 105% (w/w), such as about 0.01 to about 90, about0.01 to about 80, about 0.01 to about 50, about 0.01 to about 20, about0.01 to about 10, about 0.02 to about 50, about 0.05 to about 50, about0.1 to about 50, or 0.1 to about 20% (w/w).

In some embodiments, the isoprene composition includes one or more ofthe following: an alcohol, an aldehyde, or a ketone (such as any of thealcohols, aldehydes, or ketones described herein). In some embodiments,the isoprene composition includes (i) an alcohol and an aldehyde, (ii)an alcohol and a ketone, (iii) an aldehyde and a ketone, or (iv) analcohol, an aldehyde, and a ketone.

In some embodiments, the isoprene composition contains one or more ofthe following: methanol, acetaldehyde, ethanol, methanethiol, 1-butanol,3-methyl-1-propanol, acetone, acetic acid, 2-butanone,2-methyl-1-butanol, or indole. In some embodiments, the isoprenecomposition contains 1 ppm or more of one or more of the following:methanol, acetaldehyde, ethanol, methanethiol, 1-butanol,3-methyl-1-propanol, acetone, acetic acid, 2-butanone,2-methyl-1-butanol, or indole. In some embodiments, the concentration ofmore of one or more of the following: methanol, acetaldehyde, ethanol,methanethiol, 1-butanol, 3-methyl-1-propanol, acetone, acetic acid,2-butanone, 2-methyl-1-butanol, or indole, is between about 1 to about10,000 ppm in an isoprene composition (such as off-gas before it ispurified). In some embodiments, the isoprene composition (such asoff-gas after it has undergone one or more purification steps) includesone or more of the following: methanol, acetaldehyde, ethanol,methanethiol, 1-butanol, 3-methyl-1-propanol, acetone, acetic acid,2-butanone, 2-methyl-1-butanol, or indole, at a concentration betweenabout 1 to about 100 ppm, such as about 1 to about 10 ppm, about 10 toabout 20 ppm, about 20 to about 30 ppm, about 30 to about 40 ppm, about40 to about 50 ppm, about 50 to about 60 ppm, about 60 to about 70 ppm,about 70 to about 80 ppm, about 80 to about 90 ppm, or about 90 to about100 ppm. Volatile organic compounds from cell cultures (such as volatileorganic compounds in the headspace of cell cultures) can be analyzedusing standard methods such as those described herein or other standardmethods such as proton transfer reaction-mass spectrometry (see, forexample, Bunge et al., Applied and Environmental Microbiology,74(7):2179-2186, 2008 which is hereby incorporated by reference in itsentirety, particular with respect to the analysis of volatile organiccompounds).

In some embodiments, the composition comprises greater than about 2 mgof isoprene, such as greater than or about 5, 10, 20, 30, 40, 50, 60,70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 mg ofisoprene. In some embodiments, the composition comprises greater than orabout 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 g of isoprene. Insome embodiments, the amount of isoprene in the composition is betweenabout 2 to about 5,000 mg, such as between about 2 to about 100 mg,about 100 to about 500 mg, about 500 to about 1,000 mg, about 1,000 toabout 2,000 mg, or about 2,000 to about 5,000 mg. In some embodiments,the amount of isoprene in the composition is between about 20 to about5,000 mg, about 100 to about 5,000 mg, about 200 to about 2,000 mg,about 200 to about 1,000 mg, about 300 to about 1,000 mg, or about 400to about 1,000 mg. In some embodiments, greater than or about 20, 25,30, 40, 50, 60, 70, 80, 90, or 95% by weight of the volatile organicfraction of the composition is isoprene.

In some embodiments, the composition includes ethanol. In someembodiments, the composition includes between about 75 to about 90% byweight of ethanol, such as between about 75 to about 80%, about 80 toabout 85%, or about 85 to about 90% by weight of ethanol. In someembodiments in which the composition includes ethanol, the compositionalso includes between about 4 to about 15% by weight of isoprene, suchas between about 4 to about 8%, about 8 to about 12%, or about 12 toabout 15% by weight of isoprene.

In some embodiments described herein, a cell comprising one or moreheterologous nucleic acids encoding an isoprene synthase polypeptide,DXS polypeptide, IDI polypeptide, and/or MVA pathway polypeptideproduces an amount of an isoprenoid compound (such as a compound with 10or more carbon atoms that is formed from the reaction of one or more IPPmolecules with one or more DMAPP molecules) that is greater than orabout 2-fold, 3-fold, 5-fold, 10-fold, 25-fold, 50-fold, 100-fold,150-fold, 200-fold, 400-fold, or greater than the amount of theisoprenoid compound produced from a corresponding cell grown underessentially the same conditions without the one or more heterologousnucleic acids. In some embodiments described herein, a cell comprisingone or more heterologous nucleic acids encoding an isoprene synthasepolypeptide, DXS polypeptide, IDI polypeptide, and/or MVA pathwaypolypeptide produces an amount of a C5 prenyl alcohol (such as3-methyl-3-buten-1-ol or 3-methyl-2-buten-1-ol) that is greater than orabout 2-fold, 3-fold, 5-fold, 10-fold, 25-fold, 50-fold, 100-fold,150-fold, 200-fold, 400-fold, or greater than the amount of the C5prenyl alcohol produced from a corresponding cell grown underessentially the same conditions without the one or more heterologousnucleic acids.

Exemplary Co-Production of BioIsoprene and Hydrogen

In some embodiments, any of the isoprene-producing cells describedherein that comprise one or more heterologous nucleic acids encoding anisoprene synthase polypeptide, a DXS polypeptide, an IDI polypeptide,and/or an MVA pathway polypeptide operably linked to a promoter furthercomprise a heterologous nucleic acid also operably linked to a promoterencoding one or more hydrogenase polypeptides or one or morepolypeptides involved in the regulation or expression of hydrogenasepolypeptides (e.g., hydrogenase maturation proteins or transcriptionfactors). In some embodiments, any of the isoprene-producing cellsdescribed herein that comprise one or more heterologous nucleic acidsencoding an isoprene synthase polypeptide, a DXS polypeptide, an IDIpolypeptide, an MVA pathway polypeptide, one or more hydrogenasepolypeptides or one or more polypeptides involved in the regulation orexpression of hydrogenase polypeptides operably linked to a promoterfurther comprise a mutation or deletion inactivating one or morepolypeptides involved in the production of fermentation side products,one or more polypeptides involved in the regulation or expression ofgenes for the production of fermentation side products, or one or morepolypeptides involved in hydrogen reuptake. Such cells can co-produceisoprene and hydrogen.

In some embodiments of any of the aspects described herein, the cellsare bacterial cells, such as gram-positive bacterial cells (e.g.,Bacillus cells such as Bacillus subtilis cells or Streptomyces cellssuch as Streptomyces lividans, Streptomyces coelicolor, or Streptomycesgriseus cells). In some embodiments of any of the aspects describedherein, the cells are gram-negative bacterial cells (e.g., Escherichiacells such as Escherichia coli cells, Rhodopseudomonas sp. such asRhodopseudomonas palustris cells, Pseudomonas sp. such as Pseudomonasfluorescens cells or Pseudomonas putida cells, or Pantoea cells such asPantoea citrea cells). In some embodiments of any of the aspectsdescribed herein, the cells are fungal, cells such as filamentous fungalcells (e.g., Trichoderma cells such as Trichoderma reesei cells orAspergillus cells such as Aspergillus oryzae and Aspergillus niger) oryeast cells (e.g., Yarrowia cells such as Yarrowia lipolytica cells orSacchraomyces cells such as Saccaromyces cerevisiae).

In some embodiments of any of the aspects described herein, the isoprenesynthase polypeptide is a polypeptide from a plant such as Pueraria(e.g., Pueraria montana or Pueraria lobata) or Populus (e.g., Populustremuloides, Populus alba, Populus nigra, Populus trichocarpa, or thehybrid, Populus alba×Populus tremula).

In some embodiments of any of the aspects described herein, the cellsfurther comprise a heterologous nucleic acid encoding an IDIpolypeptide. In some embodiments of any of the aspects described herein,the cells further comprise an insertion of a copy of an endogenousnucleic acid encoding an IDI polypeptide. In some embodiments of any ofthe aspects described herein, the cells further comprise a heterologousnucleic acid encoding a DXS polypeptide. In some embodiments of any ofthe aspects described herein, the cells further comprise an insertion ofa copy of an endogenous nucleic acid encoding a DXS polypeptide. In someembodiments of any of the aspects described herein, the cells furthercomprise one or more nucleic acids encoding an IDI polypeptide and a DXSpolypeptide. In some embodiments of any of the aspects described herein,one nucleic acid encodes the isoprene synthase polypeptide, IDIpolypeptide, and DXS polypeptide. In some embodiments of any of theaspects described herein, one vector encodes the isoprene synthasepolypeptide, IDI polypeptide, and DXS polypeptide. In some embodiments,the vector comprises a selective marker or a selectable marker, such asan antibiotic resistance nucleic acid.

In some embodiments of any of the aspects described herein, the cellsfurther comprise a heterologous nucleic acid encoding an MVA pathwaypolypeptide (such as an MVA pathway polypeptide from Saccharomycescerevisia or Enterococcus faecalis). In some embodiments of any of theaspects described herein, the cells further comprise an insertion of acopy of an endogenous nucleic acid encoding an MVA pathway polypeptide(such as an MVA pathway polypeptide from Saccharomyces cerevisia orEnterococcus faecalis). In some embodiments of any of the aspectsdescribed herein, the cells comprise an isoprene synthase, DXS, and MVApathway nucleic acid. In some embodiments of any of the aspectsdescribed herein, the cells comprise an isoprene synthase nucleic acid,a DXS nucleic acid, an IDI nucleic acid, and a MVA pathway nucleic acid.

In some embodiments, the isoprene-producing cells described hereinfurther comprise a heterologous nucleic acid encoding a hydrogenasepolypeptide operably linked to a promoter. In some embodiments, thehydrogenase polypeptide comprises E. coli hydrogenase-1 (Hyd-1), E. colihydrogenase-2 (Hyd-2), E. coli hydrogenase-3 (Hyd-3), E. colihydrogenase-4 (Hyd-4), E. coli formate hydrogen lyase (FHL) complex,which produces hydrogen gas from formate and CO₂ under anaerobicconditions at acidic pH, Rhodococcus opacus MR11 hydrogenase (R. opacusHoxH), Synechosystis sp. PCC 6803 hydrogenase (Syn. PCC 6803 HoxH),Desulfovibrio gigas hydrogenase (D. gigas), and Desulfovibriodesulfuricans ATCC 7757 hydrogenase (D. desulfuricans). In someembodiments, the isoprene-producing cells further comprising aheterologous nucleic acid encoding a hydrogenase polypeptide operablylinked to a promoter further comprise E. coli hydrogenase-3 (Hyd-3), E.coli pyruvate formate lyase (pfl), and E. coli formate hydrogen lyase(FHL) complex.

In some embodiments, the hydrogenase polypeptide encodes aferredoxin-dependent hydrogenase polypeptide. In some embodiments, theferredoxin-dependent hydrogenase polypeptide comprises Clostridiumacetobutulicum hydrogenase A (HydA), which can be expressed inconjunction with one or more of: (1) Bacillus subtilis NADPH ferredoxinoxidoreductase (NFOR) or Clostridium kluyveri NADH ferredoxinoxidoreductase (RnfCDGEAB), Clostridium pasteuranium ferredoxinoxidoreductase (Fdx); (2) glyceraldehyde-6-phosphate ferredoxinoxidoreductase (GAPOR); or (3) pyruvate ferredoxin oxidoreductase (POR).In some embodiments, the ferredoxin-dependent hydrogenase polypeptideClostridium acetobutulicum hydrogenase A (HydA) is expressed with threeHydA-associated maturation enzymes (HydE, HydG, and HydF), and furtherin conjunction with one or more of: (1) Bacillus subtilis NADPHferredoxin oxidoreductase (NFOR) or Clostridium kluyveri NADH ferredoxinoxidoreductase (RnfCDGEAB), Clostridium pasteuranium ferredoxinoxidoreductase (Fdx); (2) glyceraldehyde-6-phosphate ferredoxinoxidoreductase (GAPOR); or (3) pyruvate ferredoxin oxidoreductase (POR).

In some embodiments, the hydrogenase polypeptide encodes anNADPH-dependent hydrogenase polypeptide. In some embodiments, theNADPH-dependent hydrogenase polypeptide comprises Pyrococcus furiosushydrogenase. In some embodiments, the hydrogenase polypeptide encodes anoxygen-tolerant hydrogenase. In some embodiments, the oxygen-toleranthydrogenase comprises Rubrivivax gelatinosus hydrogenase, and Ralstoniaeutropha hydrogenase.

In some embodiments, the isoprene-producing cells described hereinfurther comprise a mutation or deletion inactivating a gene involved inregulation of hydrogenase activity, such as iron-sulfur complextranscriptional regulator (iscR) (Kalim-Akhtar et al., “Deletion of iscRstimulates recombinant Clostridial Fe/Fe hydrogenase activity andH₂-accumulation in Escherichia coli BL21(DE3),” Appl. Microbiol.Biotechnol. 78:853-862 (2008), which is incorporated herein by referencein its entirety, particularly with reference to stimulation ofClostridial Fe/Fe hydrogenase activity and hydrogen accumulation in E.coli by deleting the iscR gene).

In some embodiments, the isoprene-producing cells described hereinfurther comprise a mutation or deletion inactivating a gene encoding oneor more cellular polypeptides involved in production of fermentationside products, such as lactate, acetate, pyruvate, ethanol, succinate,and glycerol. In some embodiments, the inactivated polypeptides involvedin production of fermentation side products comprise one or morepolypeptides encoding formate dehydrogenase N, alpha subunit (fdnG),formate dehydrogenase O, large subunit (fdoG), nitrate reductase (narG),formate transporter A (focA), formate transporter B (focB), pyruvateoxidase (poxB), pyruvate dehydrogenase E1 component ackA/pta (aceE),alcohol dehydrogenase (adhE), fumarate reductase membrane protein(frdC), or lactate dehydrogenase (ldhA).

In some embodiments, the isoprene-producing cells described hereinfurther comprise a mutation or deletion inactivating a gene encoding oneor more cellular polypeptides involved in the regulation or expressionof genes involved in production of fermentation side products. In someembodiments, the inactivated polypeptides involved in the regulation orexpression of genes involved in production of fermentation side productscomprise repressor of formate hydrogen lyase (hycA), fumarate reductaseregulator (fnr), acetyl-coenzyme A synthetase (acs), and formatedehydrogenase regulatory protein (hycA).

In some embodiments, the isoprene-producing cells described hereinfurther comprise a mutation or deletion inactivating a gene encoding oneor more cellular polypeptides involved in hydrogen re-uptake. In someembodiments, the inactivated polypeptides involved in hydrogen re-uptakecomprise E. coli hydrogenase-1 (Hyd-1) (hya operon) and E. colihydrogenase-2 (Hyd-2) (hyb operon).

In some embodiments of any of the aspects described herein, theheterologous isoprene synthase, DXS polypeptide, IDI polypeptide, MVApathway, hydrogenase, hydrogenase maturation or transcription factorpolypeptide or nucleic acid is operably linked to a T7 promoter, such asa T7 promoter contained in a medium or high copy plasmid. In someembodiments of any of the aspects described herein, the heterologousisoprene synthase, DXS polypeptide, IDI polypeptide, MVA pathway,hydrogenase, hydrogenase maturation or transcription factor nucleic acidis operably linked to a Trc promoter, such as a Trc promoter containedin a medium or high copy plasmid. In some embodiments of any of theaspects described herein, the heterologous isoprene synthase, DXSpolypeptide, IDI polypeptide, MVA pathway, hydrogenase, hydrogenasematuration or transcription factor nucleic acid is operably linked to aLac promoter, such as a Lac promoter contained in a low copy plasmid. Insome embodiments of any of the aspects described herein, theheterologous isoprene synthase, DXS polypeptide, IDI polypeptide, MVApathway, hydrogenase, hydrogenase maturation or transcription factornucleic acid is operably linked to an endogenous promoter, such as anendogenous alkaline serine protease promoter. In some embodiments, theheterologous isoprene synthase, DXS polypeptide, IDI polypeptide, MVApathway, hydrogenase, hydrogenase maturation or transcription factornucleic acid integrates into a chromosome of the cells without aselective marker or without a selectable marker.

In some embodiments, one or more MVA pathway, IDI, DXS, isoprenesynthase, hydrogenase, hydrogenase maturation or transcription factornucleic acids are placed under the control of a promoter or factor thatis more active in stationary phase than in the growth phase. Forexample, one or more MVA pathway, IDI, DXS, isoprene synthase,hydrogenase, hydrogenase maturation or transcription factor nucleicacids may be placed under control of a stationary phase sigma factor,such as RpoS. In some embodiments, one or more MVA pathway, IDI, DXS,isoprene synthase, hydrogenase, hydrogenase maturation or transcriptionfactor nucleic acids are placed under control of a promoter inducible instationary phase, such as a promoter inducible by a response regulatoractive in stationary phase.

In some embodiments of any of the aspects described herein, at least aportion of the cells maintain the heterologous isoprene synthase, DXSpolypeptide, IDI polypeptide, MVA pathway, hydrogenase, hydrogenasematuration or transcription factor nucleic acid for at least or about 5,10, 20, 40, 50, 60, 65, or more cell divisions in a continuous culture(such as a continuous culture without dilution). In some embodiments ofany of the aspects described herein, the nucleic acid comprising theisoprene synthase, DXS polypeptide, IDI polypeptide, MVA pathway,hydrogenase, hydrogenase maturation or transcription factor nucleic acidalso comprises a selective marker or a selectable marker, such as anantibiotic resistance nucleic acid.

In some embodiments of any of the aspects described herein, cells thatco-produce isoprene and hydrogen are cultured in any of the culturemedia described herein, under oxygen-limited conditions to facilitatethe co-production of isoprene and hydrogen by the cells. In someembodiments, the cells are grown in oxygen-limited culture. In someembodiments, the cells are grown in the presence of 0.5 moles of oxygenper mole of isoprene. In some embodiments, the cells are grownanaerobically, in the absence of oxygen.

In some embodiments, any of the cells described herein are grown inoxygen-limited culture and co-produce isoprene and hydrogen. In someembodiments, the cells in oxygen-limited culture produce isoprene at arate greater than about 400 nmole/g_(wcm)/hr, and produce hydrogen at arate greater than about 125 nmole/g_(wcm)/hr. In some embodiments, thecells in oxygen-limited culture produce isoprene at a rate between about400 nmole/g_(wcm)/hr to about 2.0×10⁵ nmole/g_(wcm)/hr and hydrogen at arate between about 125 nmole/g_(wcm)/hr to about 1.25×10⁴nmole/g_(wcm)/hr. In some embodiments, the cells in oxygen-limitedculture produce isoprene at a rate between about 400 nmole/g_(wcm)/hrand about 2.0×10⁵ nmole/g_(wcm)/hr, between about 500 nmole/g_(wcm)/hrand about 1.5×10⁵ nmole/g_(wcm)/hr, between about 750 nmole/g_(wcm)/hrand about 1×10⁵ nmole/g_(wcm)/hr, between about 1000 nmole/g_(wcm)/hrand about 1×10⁵ nmole/g_(wcm)/hr, between about 2500 nmole/g_(wcm)/hrand about 1×10⁵ nmole/g_(wcm)/hr, between about 5000 nmole/g_(wcm)/hrand about 1×10⁵ nmole/g_(wcm)/hr, between about 7500 nmole/g_(wcm)/hrand about 1×10⁵ nmole/g_(wcm)/hr, and between about 1×10⁴nmole/g_(wcm)/hr and about 1×10⁵ nmole/g_(wcm)/hr. In some embodiments,the cells in oxygen-limited culture produce greater than about 400, 500,600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000,4,000, 5,000, or more nmole/g_(wcm)/hr isoprene. In some embodiments,the cells in oxygen-limited culture produce hydrogen at a rate betweenabout 125 nmole/g_(wcm)/hr to about 1.25×10⁴ nmole/g_(wcm)/hr, betweenabout 250 nmole/g_(wcm)/hr to about 1.25×10⁴ nmole/g_(wcm)/hr, betweenabout 500 nmole/g_(wcm)/hr to about 1.25×10⁴ nmole/g_(wcm)/hr, betweenabout 750 nmole/g_(wcm)/hr to about 1.25×10⁴ nmole/g_(wcm)/hr, betweenabout 1000 nmole/g_(wcm)/hr to about 1.25×10⁴ nmole/g_(wcm)/hr, betweenabout 1250 nmole/g_(wcm)/hr to about 1.25×10⁴ nmole/g_(wcm)/hr, betweenabout 2500 nmole/g_(wcm)/hr to about 1.25×10⁴ nmole/g_(wcm)/hr, betweenabout 5000 nmole/g_(wcm)/hr to about 1.25×10⁴ nmole/g_(wcm)/hr, betweenabout 7500 nmole/g_(wcm)/hr to about 1.25×10⁴ nmole/g_(wcm)/hr, andbetween about 1.00×10⁴ nmole/g_(wcm)/hr to about 1.25×10⁴nmole/g_(wcm)/hr. In some embodiments, the cells in oxygen-limitedculture produce greater than about 125, 250, 500, 750, 1000, 1,250,1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000, 7,500, 10,000, or morenmole/g_(wcm)/hr hydrogen.

In some embodiments, any of the cells described herein are grown inoxygen-limited culture and co-produce isoprene and hydrogen. In someembodiments, the cells in oxygen-limited culture have an averagevolumetric productivity of isoprene greater than about 0.1mg/L_(broth)/hr and an average volumetric productivity of hydrogengreater than about 0.005 mg/L_(broth)/hr. In some embodiments, the cellsin oxygen-limited culture have a peak volumetric productivity ofisoprene greater than about 1000 mg/L_(broth)/hr and a peak volumetricproductivity of hydrogen greater than about 5 mg/L_(broth)/hr. In someembodiments, the cells in oxygen-limited culture have a peak volumetricproductivity of isoprene greater than about 3000 mg/L_(broth)/hr and apeak volumetric productivity of hydrogen greater than about 5mg/L_(broth)/hr. In some embodiments, the cells in oxygen-limitedculture have a peak volumetric productivity of isoprene greater thanabout 5000 mg/L_(broth)/hr and a peak volumetric productivity ofhydrogen greater than about 5 mg/L_(broth)/hr. In some embodiments, thecells in oxygen-limited culture have an average volumetric productivityof isoprene between about 0.1 mg/L_(broth)/hr and about 5000mg/L_(broth)/hr, and an average volumetric productivity of hydrogenbetween about 0.005 mg/L_(broth)/hr and about 5 mg/L_(broth)/hr. In someembodiments, the cells in oxygen-limited culture have an averagevolumetric productivity of isoprene between about 1 mg/L_(broth)/hr andabout 5000 mg/L_(broth)/hr, between about 5 mg/L_(broth)/hr and about5000 mg/L_(broth)/hr, between about 10 mg/L_(broth)/hr and about 5000mg/L_(broth)/hr, between about 25 mg/L_(broth)/hr and about 5000mg/L_(broth)/hr, between about 50 mg/L_(broth)/hr and about 5000mg/L_(broth)/hr, between about 100 mg/L_(broth)/hr and about 5000mg/L_(broth)/hr, between about 250 mg/L_(broth)/hr and about 5000mg/L_(broth)/hr, between about 500 mg/L_(broth)/hr and about 5000mg/L_(broth)/hr, between about 1000 mg/L_(broth)/hr and about 5000mg/L_(broth)/hr, and between about 2500 mg/L_(broth)/hr and about 5000mg/L_(broth)/hr, and an average volumetric productivity of hydrogenbetween about 0.01 mg/L_(broth)/hr and about 5 mg/L_(broth)/hr, betweenabout 0.025 mg/L_(broth)/hr and about 5 mg/L_(broth)/hr, between about0.05 mg/L_(broth)/hr and about 5 mg/L_(broth)/hr, between about 0.1mg/L_(broth)/hr and about 5 mg/L_(broth)/hr, between about 0.25mg/L_(broth)/hr and about 5 mg/L_(broth)/hr, between about 0.5mg/L_(broth)/hr and about 5 mg/L_(broth)/hr, between about 1mg/L_(broth)/hr and about 5 mg/L_(broth)/hr, and between about 2.5mg/L_(broth)/hr and about 5 mg/L_(broth)/hr.

In some embodiments, any of the cells described herein are grown inoxygen-limited culture and co-produce isoprene and hydrogen. In someembodiments, the cells in oxygen-limited culture convert more than about0.002 molar percent of the carbon that the cells consume from a cellculture medium into isoprene, and produce hydrogen equivalent to morethan about 0.024 molar percent of the carbon that the cells consume froma cell culture medium. In some embodiments, the cells in oxygen-limitedculture convert more than about 0.002 molar percent of the carbon thatthe cells consume from a cell culture medium into isoprene, and producehydrogen equivalent to more than about 400 molar percent of the carbonthat the cells consumer from a cell culture medium.

In some embodiments, any of the cells described herein that co-produceisoprene and hydrogen are grown in oxygen-limited culture. In someembodiments, the cells in oxygen-limited culture co-produce isoprene andhydrogen in a ratio ranging from at least one molar percent of isoprenefor every three molar percent of hydrogen to at least one molar percentof isoprene for every four molar percent of hydrogen. In someembodiments, the cells in oxygen-limited culture produce an off-gascontaining from 1 to 11 molar percent isoprene and from 3 to 33 molarpercent hydrogen. In some embodiments, the cells produce from 1 to 11molar percent isoprene and from 4 to 44 molar percent hydrogen. In someembodiments, the cells in oxygen-limited culture also produce an off-gascontaining oxygen, carbon dioxide, or nitrogen. In some embodiments, thecells in oxygen limited culture produce an off-gas containing from 0 to21 molar percent oxygen, from 18 to 44 molar percent carbon dioxide, andfrom 0 to 78 molar percent nitrogen.

In another aspect, provided herein are cells in oxygen-limited culturethat co-produce isoprene and hydrogen, comprising a heterologous nucleicacid encoding an isoprene synthase polypeptide, wherein the cells: (i)produce isoprene at a rate greater than about 400 nmole/g_(wcm)/hr andproduce hydrogen at a rate greater than about 125 nmole/g_(wcm)/hr; (ii)have an average volumetric productivity of isoprene greater than about0.1 mg/L_(broth)/hr and an average volumetric productivity of hydrogengreater than about 0.005 mg/L_(broth)/hr; or (iii) convert more thanabout 0.002 molar percent of the carbon that the cells consume from acell culture medium into isoprene, and produce hydrogen equivalent tomore than about 0.024 molar percent of the carbon that the cells consumefrom a cell culture medium.

In some embodiments, the cells in oxygen-limited culture comprise aheterologous nucleic acid encoding an isoprene synthase polypeptide,wherein the heterologous nucleic acid is operably linked to a promoter,and wherein the cells produce greater than about 400 nmole/g_(wcm)/hr ofisoprene and greater than about 125 nmole/g_(wcm)/hr of hydrogen. Insome embodiments, the cells in oxygen-limited culture comprise aheterologous nucleic acid encoding an isoprene synthase polypeptide,wherein the heterologous nucleic acid is operably linked to a promoter,and wherein the cells have an average volumetric productivity ofisoprene greater than about 0.1 mg/L_(broth)/hr and an averagevolumetric productivity of hydrogen greater than about 0.005mg/L_(broth)/hr. In some embodiments, the cells in oxygen-limitedculture comprise a heterologous nucleic acid encoding an isoprenesynthase polypeptide, wherein the heterologous nucleic acid is operablylinked to a promoter, and wherein the cells convert more than about0.002 molar percent of the carbon that the cells consume from a cellculture medium into isoprene, and more than about 0.024 molar percent ofthe carbon that the cells consume from a cell culture medium intohydrogen. In some embodiments, the isoprene synthase polypeptide is aplant isoprene synthase polypeptide.

In some embodiments, the cells in oxygen-limited culture comprising aheterologous nucleic acid encoding an isoprene synthase polypeptideproduce isoprene at a rate between about 400 nmole/g_(wcm)/hr and about2.0×10⁵ nmole/g_(wcm)/hr, between about 500 nmole/g_(wcm)/hr and about1.5×10⁵ nmole/g_(wcm)/hr, between about 750 nmole/g_(wcm)/hr and about1×10⁵ nmole/g_(wcm)/hr, between about 1000 nmole/g_(wcm)/hr and about1×10⁵ nmole/g_(wcm)/hr, between about 2500 nmole/g_(wcm)/hr and about1×10⁵ nmole/g_(wcm)/hr, between about 5000 nmole/g_(wcm)/hr and about1×10⁵ nmole/g_(wcm)/hr, between about 7500 nmole/g_(wcm)/hr and about1×10⁵ nmole/g_(wcm)/hr, and between about 1×10⁴ nmole/g_(wcm)/hr andabout 1×10⁵ nmole/g_(wcm)/hr, and produce hydrogen at a rate betweenabout 125 nmole/g_(wcm)/hr to about 1.25×10⁴ nmole/g_(wcm)/hr, betweenabout 250 nmole/g_(wcm)/hr to about 1.25×10⁴ nmole/g_(wcm)/hr, betweenabout 500 nmole/g_(wcm)/hr to about 1.25×10⁴ nmole/g_(wcm)/hr, betweenabout 750 nmole/g_(wcm)/hr to about 1.25×10⁴ nmole/g_(wcm)/hr, betweenabout 1000 nmole/g_(wcm)/hr to about 1.25×10⁴ nmole/g_(wcm)/hr, betweenabout 1250 nmole/g_(wcm)/hr to about 1.25×10⁴ nmole/g_(wcm)/hr, betweenabout 2500 nmole/g_(wcm)/hr to about 1.25×10⁴ nmole/g_(wcm)/hr, betweenabout 5000 nmole/g_(wcm)/hr to about 1.25×10⁴ nmole/g_(wcm)/hr, betweenabout 7500 nmole/g_(wcm)/hr to about 1.25×10⁴ nmole/g_(wcm)/hr, andbetween about 1.00×10⁴ nmole/g_(wcm)/hr to about 1.25×10⁴nmole/g_(wcm)/hr.

In some embodiments, provided herein are methods of co-producingisoprene and hydrogen, the methods comprising: (a) culturing cells underconditions suitable for the co-production of isoprene and hydrogen; and(b) co-producing isoprene and hydrogen, wherein the cells producegreater than about 400 nmole/g_(wcm)/hour of isoprene, and wherein thecells produce greater than about 125 nmole/g_(wcm)/hr of hydrogen.

In some embodiments, the cells in oxygen-limited culture comprising aheterologous nucleic acid encoding an isoprene synthase polypeptideproduce isoprene at a rate between about 400 nmole/g_(wcm)/hr and about2.0×10⁵ nmole/g_(wcm)/hr, between about 500 nmole/g_(wcm)/hr and about1.5×10⁵ nmole/g_(wcm)/hr, between about 750 nmole/g_(wcm)/hr and about1×10⁵ nmole/g_(wcm)/hr, between about 1000 nmole/g_(wcm)/hr and about1×10⁵ nmole/g_(wcm)/hr, between about 2500 nmole/g_(wcm)/hr and about1×10⁵ nmole/g_(wcm)/hr, between about 5000 nmole/g_(wcm)/hr and about1×10⁵ nmole/g_(wcm)/hr, between about 7500 nmole/g_(wcm)/hr and about1×10⁵ nmole/g_(wcm)/hr, and between about 1×10⁴ nmole/g_(wcm)/hr andabout 1×10⁵ nmole/g_(wcm)/hr, and produce hydrogen at a rate betweenabout 125 nmole/g_(wcm)/hr to about 1.25×10⁴ nmole/g_(wcm)/hr, betweenabout 250 nmole/g_(wcm)/hr to about 1.25×10⁴ nmole/g_(wcm)/hr, betweenabout 500 nmole/g_(wcm)/hr to about 1.25×10⁴ nmole/g_(wcm)/hr, betweenabout 750 nmole/g_(wcm)/hr to about 1.25×10⁴ nmole/g_(wcm)/hr, betweenabout 1000 nmole/g_(wcm)/hr to about 1.25×10⁴ nmole/g_(wcm)/hr, betweenabout 1250 nmole/g_(wcm)/hr to about 1.25×10⁴ nmole/g_(wcm)/hr, betweenabout 2500 nmole/g_(wcm)/hr to about 1.25×10⁴ nmole/g_(wcm)/hr, betweenabout 5000 nmole/g_(wcm)/hr to about 1.25×10⁴ nmole/g_(wcm)/hr, betweenabout 7500 nmole/g_(wcm)/hr to about 1.25×10⁴ nmole/g_(wcm)/hr, andbetween about 1.00×10⁴ nmole/g_(wcm)/hr to about 1.25×10⁴nmole/g_(wcm)/hr.

In some embodiments, provided herein are methods of co-producingisoprene and hydrogen, the methods comprising: (a) culturing cells underconditions suitable for the co-production of isoprene and hydrogen; and(b) co-producing isoprene and hydrogen, wherein the cells have anaverage volumetric productivity of isoprene greater than about 0.1mg/L_(broth)/hr and an average volumetric productivity of hydrogengreater than about 0.005 mg/L_(broth)/hr.

In some embodiments, provided herein are methods of co-producingisoprene and hydrogen, the methods comprising: (a) culturing cells underconditions suitable for the co-production of isoprene and hydrogen; and(b) co-producing isoprene and hydrogen, wherein the cells convert morethan about 0.002 molar percent of the carbon that the cells consume froma cell culture medium into isoprene, and produce hydrogen equivalent tomore than about 0.024 molar percent of the carbon that the cells consumefrom a cell culture medium.

In some embodiments, provided herein are compositions comprisingisoprene and hydrogen in a ratio ranging from at least one molar percentof isoprene for every three molar percent of hydrogen to at least onemolar percent of isoprene for every four molar percent of hydrogen, and0.1 molar percent or less of volatile impurities. In some embodiments,the compositions further comprise from 1 to 11 molar percent isopreneand from 4 to 44 molar percent hydrogen. In some embodiments, thecompositions further comprise oxygen, carbon dioxide, or nitrogen. Insome embodiments, the compositions further comprise from 0 to 21 molarpercent oxygen, from 18 to 44 molar percent carbon dioxide, and from 0to 78 molar percent nitrogen. In some embodiments, the compositionfurther comprises 1.0×10⁻⁴ molar percent or less of non-methane volatileimpurities. In some embodiments, the non-methane volatile impuritiescomprise one or more of the following: 2-heptanone,6-methyl-5-hepten-2-one, 2,4,5-trimethylpyridine,2,3,5-trimethylpyrazine, citronellal, acetaldehyde, methanethiol, methylacetate, 1-propanol, diacetyl, 2-butanone, 2-methyl-3-buten-2-ol, ethylacetate, 2-methyl-1-propanol, 3-methyl-1-butanal, 3-methyl-2-butanone,1-butanol, 2-pentanone, 3-methyl-1-butanol, ethyl isobutyrate,3-methyl-2-butenal, butyl acetate, 3-methylbutyl acetate,3-methyl-3-buten-1-yl acetate, 3-methyl-2-buten-1-yl acetate,3-hexen-1-ol, 3-hexen-1-yl acetate, limonene, geraniol(trans-3,7-dimethyl-2,6-octadien-1-ol), citronellol(3,7-dimethyl-6-octen-1-ol), (E)-3,7-dimethyl-1,3,6-octatriene,(Z)-3,7-dimethyl-1,3,6-octatriene, 2,3-cycloheptenolpyridine, or alinear isoprene polymer (such as a linear isoprene dimer or a linearisoprene trimer derived from the polymerization of multiple isopreneunits). In some embodiments, the non-methane volatile impuritiescomprise one or more of the following: the isoprene composition includesone or more of the following: an alcohol, an aldehyde, or a ketone (suchas any of the alcohols, aldehydes, or ketones described herein). In someembodiments, the isoprene composition includes (i) an alcohol and analdehyde, (ii) an alcohol and a ketone, (iii) an aldehyde and a ketone,or (iv) an alcohol, an aldehyde, and a ketone. In some embodiments, thenon-methane volatile impurities comprise one or more of the following:methanol, acetaldehyde, ethanol, methanethiol, 1-butanol,3-methyl-1-propanol, acetone, acetic acid, 2-butanone,2-methyl-1-butanol, or indole.

Also provided herein are methods of co-producing isoprene and hydrogen,the methods comprising: a) culturing cells under conditions suitable forthe co-production of isoprene and hydrogen; and b) co-producing isopreneand hydrogen, wherein the peak concentration of the isoprene produced bythe cells in oxygen-limited culture is greater than about 10ng/L_(broth) and the hydrogen evolution rate of the cells is greaterthan about 0.0025 mmol/L_(broth)/hour. In some embodiments of any ofthese methods, the hydrogen evolution rate is between about any of0.0025 mmol/L_(broth)/hr and about 10 mmol/L_(broth)/hr, between about0.0025 mmol/L_(broth)/hr and about 5 mmol/L_(broth)/hr, between about0.0025 mmol/L_(broth)/hr and about 2.5 mmol/L_(broth)/hr, between about0.0025 mmol/L_(broth)/hr and about 1 mmol/L_(broth)/hr, between about0.0025 mmol/L_(broth)/hr and about 0.5 mmol/L_(broth)/hr, between about0.0025 mmol/L_(broth)/hr and about 0.25 mmol/L_(broth)/hr, between about0.0025 mmol/L_(broth)/hr and about 0.025 mmol/L_(broth)/hr, betweenabout 0.025 mmol/L_(broth)/hr and about 0.5 mmol/L_(broth)/hr, betweenabout 0.025 mmol/L_(broth)/hr and about 1 mmol/L_(broth)/hr, betweenabout 0.025 mmol/L_(broth)/hr and about 2.5 mmol/L_(broth)/hr, betweenabout 0.025 mmol/L_(broth)/hr and about 5 mmol/L_(broth)/hr, betweenabout 0.025 mmol/L_(broth)/hr and about 10 mmol/L_(broth)/hr, betweenabout 0.25 mmol/L_(broth)/hr and 1 mmol/L_(broth)/hr, between about 0.25mmol/L_(broth)/hr and 2.5 mmol/L_(broth)/hr, between about 0.25mmol/L_(broth)/hr and 2.5 mmol/L_(broth)/hr, and between about 0.25mmol/L_(broth)/hr and 10 mmol/L_(broth)/hr.

Provided herein are also methods of co-producing isoprene and hydrogencomprising a) culturing cells under conditions suitable for theco-production of isoprene and hydrogen; and b) co-producing isoprene andhydrogen, wherein the liquid phase concentration of isoprene is lessthan about 200 mg/L, the cells produce greater than about 400nmole/g_(wcm)/hour of isoprene, and the hydrogen evolution rate of thecells is greater than about 0.0025 mmol/L/hour. In some embodiments, theliquid phase concentration of isoprene in the culture is less than aboutany of 175 mg/L, 150 mg/L, 125 mg/L, 100 mg/L, 75 mg/L, 50 mg/L, 25mg/L, 20 mg/L, 15 mg/L, 10 mg/L, 5 mg/L, or 2.5 mg/L. In someembodiments, the liquid phase concentration of isoprene in culture isbetween about any of 0.1 mg/L to 200 mg/L, 1 mg/L to 200 mg/L, 1 mg/L to150 mg/L, 1 mg/L to 100 mg/L, 1 mg/L to 50 mg/L, 1 mg/L to 25 mg/L, 1mg/L to 20 mg/L, or 10 mg/L to 20 mg/L. In some embodiments of any ofthese methods, the hydrogen evolution rate is between about any of0.0025 mmol/L_(broth)/hr and about 10 mmol/L_(broth)/hr, between about0.0025 mmol/L_(broth)/hr and about 5 mmol/L_(broth)/hr, between about0.0025 mmol/L_(broth)/hr and about 2.5 mmol/L_(broth)/hr, between about0.0025 mmol/L_(broth)/hr and about 1 mmol/L_(broth)/hr, between about0.0025 mmol/L_(broth)/hr and about 0.5 mmol/L_(broth)/hr, between about0.0025 mmol/L_(broth)/hr and about 0.25 mmol/L_(broth)/hr, between about0.0025 mmol/L_(broth)/hr and about 0.025 mmol/L_(broth)/hr, betweenabout 0.025 mmol/L_(broth)/hr and about 0.5 mmol/L_(broth)/hr, betweenabout 0.025 mmol/L_(broth)/hr and about 1 mmol/L_(broth)/hr, betweenabout 0.025 mmol/L_(broth)/hr and about 2.5 mmol/L_(broth)/hr, betweenabout 0.025 mmol/L_(broth)/hr and about 5 mmol/L_(broth)/hr, betweenabout 0.025 mmol/L_(broth)/hr and about 10 mmol/L_(broth)/hr, betweenabout 0.25 mmol/L_(broth)/hr and 1 mmol/L_(broth)/hr, between about 0.25mmol/L_(broth)/hr and 2.5 mmol/L_(broth)/hr, between about 0.25mmol/L_(broth)/hr and 2.5 mmol/L_(broth)/hr, and between about 0.25mmol/L_(broth)/hr and 10 mmol/L_(broth)/hr.

In one aspect, provided herein are cells in oxygen-limited culture thatco-produce isoprene and hydrogen. In some embodiments, theoxygen-limited culture is anaerobic. In some embodiments, the cells inoxygen-limited culture produce greater than about 400 nmole/g_(wcm)/hrof isoprene and greater than about 125 nmole/g_(wcm)/hr of hydrogen. Insome embodiments, the cells have a heterologous nucleic acid that (i)encodes an isoprene synthase polypeptide and (ii) is operably linked toa promoter. In some embodiments, the cells are cultured in a culturemedium that includes a carbon source, such as, but not limited to, acarbohydrate, glycerol, glycerine, dihydroxyacetone, one-carbon source,oil, animal fat, animal oil, fatty acid, lipid, phospholipid,glycerolipid, monoglyceride, diglyceride, triglyceride, renewable carbonsource, polypeptide (e.g., a microbial or plant protein or peptide),yeast extract, component from a yeast extract, or any combination of twoor more of the foregoing. In some embodiments, the cells are culturedunder limited glucose conditions.

In some embodiments, provided herein are cells in oxygen-limited culturethat convert more than about 0.002% of the carbon in a cell culturemedium into isoprene and produce hydrogen equivalent to more than about0.024 molar percent of the carbon in a cell culture medium. In someembodiments, the oxygen-limited culture is anaerobic. In someembodiments, the cells have a heterologous nucleic acid that (i) encodesan isoprene synthase polypeptide and (ii) is operably linked to apromoter. In some embodiments, the cells are cultured in a culturemedium that includes a carbon source, such as, but not limited to, acarbohydrate, glycerol, glycerine, dihydroxyacetone, one-carbon source,oil, animal fat, animal oil, fatty acid, lipid, phospholipid,glycerolipid, monoglyceride, diglyceride, triglyceride, renewable carbonsource, polypeptide (e.g., a microbial or plant protein or peptide),yeast extract, component from a yeast extract, or any combination of twoor more of the foregoing. In some embodiments, the cells are culturedunder limited glucose conditions.

In some embodiments, provided herein are cells in oxygen-limited culturethat comprise a heterologous nucleic acid encoding an isoprene synthasepolypeptide. In some embodiments, the oxygen-limited culture isanaerobic. In some embodiments, the cells have a heterologous nucleicacid that (i) encodes an isoprene synthase polypeptide and (ii) isoperably linked to a promoter. In some embodiments, the cells arecultured in a culture medium that includes a carbon source, such as, butnot limited to, a carbohydrate, glycerol, glycerine, dihydroxyacetone,one-carbon source, oil, animal fat, animal oil, fatty acid, lipid,phospholipid, glycerolipid, monoglyceride, diglyceride, triglyceride,renewable carbon source, polypeptide (e.g., a microbial or plant proteinor peptide), yeast extract, component from a yeast extract, or anycombination of two or more of the foregoing. In some embodiments, thecells are cultured under limited glucose conditions.

In one aspect, provided herein are methods of co-producing isoprene withanother compound, such as methods of using any of the cells describedherein to co-produce isoprene and hydrogen. In some embodiments, themethod involves culturing cells under oxygen-limited conditionssufficient to produce greater than about 400 nmole/g_(wcm)/hr ofisoprene and greater than about 125 nmole/g_(wcm)/hr of hydrogen. Insome embodiments, the oxygen-limited culture is anaerobic. In someembodiments, the method also includes recovering the isoprene andhydrogen produced by the cells. In some embodiments, the method furtherincludes purifying the isoprene and the hydrogen produced by the cells.In some embodiments, the method includes polymerizing the isoprene. Insome embodiments, the cells have a heterologous nucleic acid that (i)encodes an isoprene synthase polypeptide and (ii) is operably linked toa promoter. In some embodiments, the cells are cultured in a culturemedium that includes a carbon source, such as, but not limited to, acarbohydrate, glycerol, glycerine, dihydroxyacetone, one-carbon source,oil, animal fat, animal oil, fatty acid, lipid, phospholipid,glycerolipid, monoglyceride, diglyceride, triglyceride, renewable carbonsource, polypeptide (e.g., a microbial or plant protein or peptide),yeast extract, component from a yeast extract, or any combination of twoor more of the foregoing. In some embodiments, the cells are culturedunder limited glucose conditions. In various embodiments, the amount ofisoprene produced (such as the total amount of isoprene produced or theamount of isoprene produced per liter of broth per hour per OD₆₀₀)during stationary phase is greater than or about 2 or more times theamount of isoprene produced during the growth phase for the same lengthof time.

In some embodiments, the method includes culturing cells underoxygen-limited conditions sufficient to convert more than about 0.002%of the carbon (mol/mol) in a cell culture medium into isoprene and toproduce hydrogen equivalent to more than about 0.024 molar percent ofthe carbon in a cell culture medium. In some embodiments, theoxygen-limited culture is anaerobic. In some embodiments, the methodalso includes recovering isoprene and hydrogen produced by the cells. Insome embodiments, the method further includes purifying isoprene andhydrogen produced by the cells. In some embodiments, the method includespolymerizing the isoprene. In some embodiments, the cells have aheterologous nucleic acid that (i) encodes an isoprene synthasepolypeptide and (ii) is operably linked to a promoter. In someembodiments, the cells are cultured in a culture medium that includes acarbon source, such as, but not limited to, a carbohydrate, glycerol,glycerine, dihydroxyacetone, one-carbon source, oil, animal fat, animaloil, fatty acid, lipid, phospholipid, glycerolipid, monoglyceride,diglyceride, triglyceride, renewable carbon source, polypeptide (e.g., amicrobial or plant protein or peptide), yeast extract, component from ayeast extract, or any combination of two or more of the foregoing.

In some embodiments of any of the aspects described herein, themicrobial polypeptide carbon source includes one or more polypeptidesfrom yeast or bacteria. In some embodiments of any of the aspectsdescribed herein, the plant polypeptide carbon source includes one ormore polypeptides from soy, corn, canola, jatropha, palm, peanut,sunflower, coconut, mustard, rapeseed, cottonseed, palm kernel, olive,safflower, sesame, or linseed.

In some embodiments, isoprene and hydrogen are only co-produced instationary phase. In some embodiments, isoprene and hydrogen areco-produced in both the growth phase and stationary phase. In variousembodiments, the amount of isoprene produced (such as the total amountof isoprene produced or the amount of isoprene produced per liter ofbroth per hour per OD₆₀₀) during stationary phase is greater than orabout 2, 3, 4, 5, 10, 20, 30, 40, 50, or more times the amount ofisoprene produced during the growth phase for the same length of time.In various embodiments, the amount of hydrogen produced (such as thetotal amount of hydrogen produced or the amount of hydrogen produced perliter of broth per hour per OD₆₀₀) during stationary phase is greaterthan or about 2, 3, 4, 5, 10, 20, 30, 40, 50, or more times the amountof hydrogen produced during the growth phase for the same length oftime.

In some embodiments, the compositions provided herein comprise hydrogenand greater than or about 99.90, 99.92, 99.94, 99.96, 99.98, or 100%isoprene by weight compared to the total weight of all C5 hydrocarbonsin the composition. In some embodiments, the composition comprises lessthan or about 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001,0.0005, 0.0001, 0.00005, or 0.00001% C5 hydrocarbons other than isoprene(such as 1,3-cyclopentadiene, cis-1,3-pentadiene, trans-1,3-pentadiene,1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene, 2-methyl-1-butene,3-methyl-1-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, orcis-pent-3-ene-1-yne) by weight compared to the total weight of all C5hydrocarbons in the composition. In some embodiments, the compositionhas less than or about 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005,0.001, 0.0005, 0.0001, 0.00005, or 0.00001% for 1,3-cyclopentadiene,cis-1,3-pentadiene, trans-1,3-pentadiene, 1,4-pentadiene, 1-pentyne,2-pentyne, 1-pentene, 2-methyl-1-butene, 3-methyl-1-butyne,pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne byweight compared to the total weight of all C5 hydrocarbons in thecomposition. In particular embodiments, the composition has greater thanabout 2 mg of isoprene and has greater than or about 99.90, 99.92,99.94, 99.96, 99.98, or 100% isoprene by weight compared to the totalweight of all C5 hydrocarbons in the composition. In some embodiments,the composition has less than or about 50, 40, 30, 20, 10, 5, 1, 0.5,0.1, 0.05, 0.01, or 0.005 μg/L of a compound that inhibits thepolymerization of isoprene for any compound in the composition thatinhibits the polymerization of isoprene. In particular embodiments, thecomposition also comprises greater than about 2 mg of isoprene andgreater than about 0.48 mg of hydrogen.

In some embodiments, the volatile organic fraction of the gas phase hasless than or about 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or0.005 μg/L of a compound that inhibits the polymerization of isoprenefor any compound in the volatile organic fraction of the gas phase thatinhibits the polymerization of isoprene. In particular embodiments, thevolatile organic fraction of the gas phase also has greater than about 2mg of isoprene and greater than about 0.48 mg of hydrogen.

In some embodiments, the systems include any of the cells and/orcompositions described herein. In some embodiments, the system includesa reactor that chamber comprises cells in oxygen-limited culture thatproduce greater than about 400, 500, 600, 700, 800, 900, 1,000, 1,250,1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000, or morenmole/g_(wcm)/hr isoprene and greater than about 125, 250, 500, 750,1000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000, 7,500,10,000, or more nmole/g_(wcm)/hr hydrogen. In some embodiments, thesystem is not a closed system. In some embodiments, at least a portionof the isoprene is removed from the system. In some embodiments, thesystem includes a gas phase comprising isoprene and hydrogen. In variousembodiments, the gas phase comprises any of the compositions describedherein.

In one aspect, featured herein is a product produced by any of thecompositions or methods described herein.

Cell Viability at High Isoprene Titer

Isoprene is a hydrophobic molecule secreted by many plants, animals, andmicrobes. Bacteria, such as Bacillus, produce isoprene at fairly lowlevels. While there is some evidence that plants secrete isoprene tohelp with thermoprotection, it has been hypothesized that isoprene mayact antagonistically to cyanobacteria or fungi, or as an antimicrobialagent. See, e.g., Ladygina et al., Process Biochemistry 41:1001-1014(2006), which is incorporated by reference in its entirety, particularlywith respect to isoprene acting antagonistically. Since the very lowproduction levels happening in nature are sufficient to beanti-microbial, it was of great concern that the titers and productivitylevels of isoprene necessary for commercialization of isoprene wouldkill the host microbe.

We have found methods for producing titers and productivity levels ofisoprene for commercialization of isoprene while maintaining cellviability and/or metabolic activity as indicated by carbon dioxideevolution rate or total carbon dioxide evolution rate.

Provided herein are methods of producing isoprene comprising: a)culturing cells under suitable conditions for production of isoprene;and b) producing isoprene, wherein cells produce greater than about 400nmole/g_(wcm)/hour of isoprene, and the carbon dioxide evolution rate ofthe cells is greater than about 1×10⁻¹⁸ mmol/L/hour. In someembodiments, the isoprene produced is any concentration or amountdisclosed in the section entitled “Exemplary Production of Isoprene.” Insome embodiments, the amount of isoprene is between about any of 400nmole/g_(wcm)/hour to 1 mole/g_(wcm)/hour, 400 nmole/g_(wcm)/hour to 1mmole/g_(wcm)/hour, 400 nmole/g_(wcm)/hour to 40 mmole/g_(wcm)/hour, 400nmole/g_(wcm)/hour to 4 mmole/g_(wcm)/hour, 1 mmole/g_(wcm)/hour to 1.5mmole/g_(wcm)/hour, 1.5 mmole/g_(wcm)/hour to 3 mmole/g_(wcm)/hour, 3mmole/g_(wcm)/hour to 5 mmole/g_(wcm)/hour, 5 mmole/g_(wcm)/hour to 25mmole/g_(wcm)/hour, 25 mmole/g_(wcm)/hour to 100 mmole/g_(wcm)/hour, 100mmole/g_(wcm)/hour to 500 mmole/g_(wcm)/hour, or 500 mmole/g_(wcm)/hourto 1000 mmole/g_(wcm)/hour. In some embodiments, the amount of isopreneis about any of 1 mmole/g_(wcm)/hour, 1.5 mmole/g_(wcm)/hour, 2mmole/g_(wcm)/hour, 3 mmole/g_(wcm)/hour, 4 mmole/g_(wcm)/hour, or 5mmole/g_(wcm)/hour. In some embodiments, the carbon dioxide evolutionrate is between about any of 1×10⁻¹⁸ mmol/L/hour to about 1 mol/L/hour,1 mmol/L/hour to 1 mol/L/hour, 25 mmol/L/hour to 750 mmol/L/hour, 25mmol/L/hour to 75 mmol/L/hour, 250 mmol/L/hour to 750 mmol/L/hour, or450 mmol/L/hour to 550 mmol/L/hour. In some embodiments, the carbondioxide evolution rate is about any of 50 mmol/L/hour, 100 mmol/L/hour,150 mmol/L/hour, 200 mmol/L/hour, 250 mmol/L/hour, 300 mmol/L/hour, 350mmol/L/hour, 400 mmol/L/hour, 450 mmol/L/hour, or 500 mmol/L/hour.

Provided herein are also methods of producing isoprene comprising: a)culturing cells under suitable conditions for production of isoprene;and b) producing isoprene, wherein cells produce greater than about 400nmole/g_(wcm)/hour of isoprene, and cell viability is reduced by lessthan about two-fold. In some embodiments, the isoprene produced is anyconcentration or amount disclosed in the section entitled “ExemplaryProduction of Isoprene.” In some embodiments, the amount of isoprene isbetween about any of 400 nmole/g_(wcm)/hour to 1 mole/g_(wcm)/hour, 400nmole/g_(wcm)/hour to 1 mmole/g_(wcm)/hour, 400 nmole/g_(wcm)/hour to 40mmole/g_(wcm)/hour, 400 nmole/g_(wcm)/hour to 4 mmole/g_(wcm)/hour, 1mmole/g_(wcm)/hour to 1.5 mmole/g_(wcm)/hour, 1.5 mmole/g_(wcm)/hour to3 mmole/g_(wcm)/hour, 3 mmole/g_(wcm)/hour to 5 mmole/g_(wcm)/hour, 5mmole/g_(wcm)/hour to 25 mmole/g_(wcm)/hour, 25 mmole/g_(wcm)/hour to100 mmole/g_(wcm)/hour, 100 mmole/g_(wcm)/hour to 500mmole/g_(wcm)/hour, or 500 mmole/g_(wcm)/hour to 1000mmole/g_(wcm)/hour. In some embodiments, the amount of isoprene is aboutany of 1 mmole/g_(wcm)/hour, 1.5 mmole/g_(wcm)/hour, 2mmole/g_(wcm)/hour, 3 mmole/g_(wcm)/hour, 4 mmole/g_(wcm)/hour, or 5mmole/g_(wcm)/hour. In some embodiments, cell viability is reduced byless than about any of 1.75-fold, 1.5-fold, 1.25-fold, 1-fold,0.75-fold, 0.5-fold, or 0.25-fold. In some embodiments, cell viabilityis reduced by about 2-fold.

Further provided herein are methods of producing isoprene comprising: a)culturing cells under suitable conditions for production of isoprene;and b) producing isoprene, wherein the cumulative total productivity ofthe isoprene produced by the cells in culture is greater than about 0.2mg/L_(broth)/hour and the carbon dioxide evolution rate of the cells isgreater than about 1×10⁻¹⁸ mmol/L/hour. In some embodiments, thecumulative total productivity of isoprene is any concentration or amountdisclosed in the section entitled “Exemplary Production of Isoprene.” Insome embodiments, the cumulative total productivity of the isoprene isbetween about any of 0.2 mg/L_(broth)/hour to 5 g/L_(broth)/hour, 0.2mg/L_(broth)/hour to 1 g/L_(broth)/hour, 1 g/L_(broth)/hour to 2.5g/L_(broth)/hour, 2.5 g/L_(broth)/hour to 5 g/L_(broth)/hour. In someembodiments, the carbon dioxide evolution rate is between about any of1×10⁻¹⁸ mmol/L/hour to about 1 mol/L/hour, 1 mmol/L/hour to 1mol/L/hour, 25 mmol/L/hour to 750 mmol/L/hour, 25 mmol/L/hour to 75mmol/L/hour, 250 mmol/L/hour to 750 mmol/L/hour, or 450 mmol/L/hour to550 mmol/L/hour. In some embodiments, the carbon dioxide evolution rateis about any of 50 mmol/L/hour, 100 mmol/L/hour, 150 mmol/L/hour, 200mmol/L/hour, 250 mmol/L/hour, 300 mmol/L/hour, 350 mmol/L/hour, 400mmol/L/hour, 450 mmol/L/hour, or 500 mmol/L/hour.

Provided herein are methods of producing isoprene comprising: a)culturing cells under suitable conditions for production of isoprene;and b) producing isoprene, wherein the cumulative total productivity ofthe isoprene produced by the cells in culture is greater than about 0.2mg/L_(broth)/hour and cell viability is reduced by less than abouttwo-fold. In some embodiments, the cumulative total productivity ofisoprene is any concentration or amount disclosed in the sectionentitled “Exemplary Production of Isoprene.” In some embodiments, thecumulative total productivity of the isoprene is between about any of0.2 mg/L_(broth)/hour to 5 g/L_(broth)/hour, 0.2 mg/L_(broth)/hour to 1g/L_(broth)/hour, 1 g/L_(broth)/hour to 2.5 g/L_(broth)/hour, 2.5g/L_(broth)/hour to 5 g/L_(broth)/hour. In some embodiments, cellviability is reduced by less than about any of 1.75-fold, 1.5-fold,1.25-fold, 1-fold, 0.75-fold, 0.5-fold, or 0.25-fold.

Methods of producing isoprene are also provided herein comprising: a)culturing cells under suitable conditions for production of isoprene;and b) producing isoprene, wherein the peak concentration of theisoprene produced by the cells in culture is greater than about 10ng/L_(broth) and the carbon dioxide evolution rate of the cells isgreater than about 1×10⁻¹⁸ mmol/L/hour. In some embodiments, the peakconcentration of isoprene is any concentration or amount disclosed inthe section entitled “Exemplary Production of Isoprene.” In someembodiments, the peak concentration of isoprene is between about any of10 ng/L_(broth) to 500 ng/L_(broth), 500 ng/L_(broth) to 1 μg/L_(broth),1 μg/L_(broth) to 5 μg/L_(broth), 5 μg/L_(broth) to 50 μg/L_(broth), 5μg/L_(broth) to 100 μg/L_(broth), 5 μg/L_(broth) to 250 μg/L_(broth),250 μg/L_(broth) to 500 μg/L_(broth), 500 μg/L_(broth) to 1mg/L_(broth), 1 mg/L_(broth) to 50 mg/L_(broth), 1 mg/L_(broth) to 100mg/L_(broth), 1 mg/L_(broth) to 200 mg/L_(broth), 10 ng/L_(broth) to 200mg/L_(broth), 5 μg/L_(broth) to 100 mg/L_(broth), or 5 μg/L_(broth) to200 mg/L_(broth). In some embodiments, the peak concentration is any ofabout 10 ng/L_(broth), 100 ng/L_(broth), 1 μg/L_(broth), 5 μg/L_(broth),1 mg/L_(broth), 30 mg/L_(broth), 100 mg/L_(broth), or 200 mg/L_(broth).In some embodiments, the carbon dioxide evolution rate is between aboutany of 1×10⁻¹⁸ mmol/L/hour to about 1 mol/L/hour, 1 mmol/L/hour to 1mol/L/hour, 25 mmol/L/hour to 750 mmol/L/hour, 25 mmol/L/hour to 75mmol/L/hour, 250 mmol/L/hour to 750 mmol/L/hour, or 450 mmol/L/hour to550 mmol/L/hour. In some embodiments, the carbon dioxide evolution rateis about any of 50 mmol/L/hour, 100 mmol/L/hour, 150 mmol/L/hour, 200mmol/L/hour, 250 mmol/L/hour, 300 mmol/L/hour, 350 mmol/L/hour, 400mmol/L/hour, 450 mmol/L/hour, or 500 mmol/L/hour.

In addition, methods of producing isoprene are also provided hereincomprising: a) culturing cells under suitable conditions for productionof isoprene; and b) producing isoprene, wherein the peak concentrationof the isoprene produced by the cells in culture is greater than about10 ng/L_(broth) and cell viability is reduced by less than abouttwo-fold. In some embodiments, the peak concentration of isoprene is anyconcentration or amount disclosed in the section entitled “ExemplaryProduction of Isoprene.” In some embodiments, the peak concentration ofisoprene is between about any of 10 ng/L_(broth) to 500 ng/L_(broth),500 ng/L_(broth) to 1 μg/L_(broth), 1 μg/L_(broth) to 5 μg/L_(broth), 5μg/L_(broth) to 50 μg/L_(broth), 5 μg/L_(broth) to 100 μg/L_(broth), 5μg/L_(broth) to 250 μg/L_(broth), 250 μg/L_(broth) to 500 μg/L_(broth),500 μg/L_(broth) to 1 mg/L_(broth), 1 mg/L_(broth) to 50 mg/L_(broth), 1mg/L_(broth) to 100 mg/L_(broth), 1 mg/L_(broth) to 200 mg/L_(broth), 10ng/L_(broth) to 200 mg/L_(broth), 5 μg/L_(broth) to 100 mg/L_(broth), or5 μg/L_(broth) to 200 mg/L_(broth). In some embodiments, the peakconcentration is any of about 10 ng/L_(broth), 100 ng/L_(broth), 1μg/L_(broth), 5 μg/L_(broth), 1 mg/L_(broth), 30 mg/L_(broth), 100mg/L_(broth), or 200 mg/L_(broth). In some embodiments, cell viabilityis reduced by less than about any of 1.75-fold, 1.5-fold, 1.25-fold,1-fold, 0.75-fold, 0.5-fold, or 0.25-fold. In some embodiments, cellviability is reduced by about 2-fold.

Cells in culture are also provided herein comprising a nucleic acidencoding an isoprene synthase polypeptide, wherein the cells producegreater than about 400 nmole/g_(wcm)/hour of isoprene and carbon dioxideevolution rate of the cells is greater than about 1×10⁻¹⁸ mmol/L/hour.In some embodiments, the isoprene produced is any concentration oramount disclosed in the section entitled “Exemplary Production ofIsoprene.” In some embodiments, the amount of isoprene is between aboutany of 400 nmole/g_(wcm)/hour to 1 mole/g_(wcm)/hour, 400nmole/g_(wcm)/hour to 1 mmole/g_(wcm)/hour, 400 nmole/g_(wcm)/hour to 40mmole/g_(wcm)/hour, 400 nmole/g_(wcm)/hour to 4 mmole/g_(wcm)/hour, 1mmole/g_(wcm)/hour to 1.5 mmole/g_(wcm)/hour, 1.5 mmole/g_(wcm)/hour to3 mmole/g_(wcm)/hour, 3 mmole/g_(wcm)/hour to 5 mmole/g_(wcm)/hour, 5mmole/g_(wcm)/hour to 25 mmole/g_(wcm)/hour, 25 mmole/g_(wcm)/hour to100 mmole/g_(wcm)/hour, 100 mmole/g_(wcm)/hour to 500mmole/g_(wcm)/hour, or 500 mmole/g_(wcm)/hour to 1000mmole/g_(wcm)/hour. In some embodiments, the amount of isoprene is aboutany of 1 mmole/g_(wcm)/hour, 1.5 mmole/g_(wcm)/hour, 2mmole/g_(wcm)/hour, 3 mmole/g_(wcm)/hour, 4 mmole/g_(wcm)/hour, or 5mmole/g_(wcm)/hour. In some embodiments, the carbon dioxide evolutionrate is between about any of 1×10⁻¹⁸ mmol/L/hour to about 1 mol/L/hour,1 mmol/L/hour to 1 mol/L/hour, 25 mmol/L/hour to 750 mmol/L/hour, 25mmol/L/hour to 75 mmol/L/hour, 250 mmol/L/hour to 750 mmol/L/hour, or450 mmol/L/hour to 550 mmol/L/hour. In some embodiments, the carbondioxide evolution rate is about any of 50 mmol/L/hour, 100 mmol/L/hour,150 mmol/L/hour, 200 mmol/L/hour, 250 mmol/L/hour, 300 mmol/L/hour, 350mmol/L/hour, 400 mmol/L/hour, 450 mmol/L/hour, or 500 mmol/L/hour.

Provided herein are also cells in culture comprising a nucleic acidencoding an isoprene synthase polypeptide, wherein cumulative totalproductivity of the isoprene produced by the cells in culture is greaterthan about 0.2 mg/L_(broth)/hour and carbon dioxide evolution rate ofthe cells is greater than about 1×10⁻¹⁸ mmol/L/hour. In someembodiments, the cumulative total productivity of isoprene is anyconcentration or amount disclosed in the section entitled “ExemplaryProduction of Isoprene.” In some embodiments, the cumulative totalproductivity of the isoprene is between about any of 0.2mg/L_(broth)/hour to 5 g/L_(broth)/hour, 0.2 mg/L_(broth)/hour to 1g/L_(broth)/hour, 1 g/L_(broth)/hour to 2.5 g/L_(broth)/hour, 2.5g/L_(broth)/hour to 5 g/L_(broth)/hour. In some embodiments, the carbondioxide evolution rate is between about any of 1×10⁻¹⁸ mmol/L/hour toabout 1 mol/L/hour, 1 mmol/L/hour to 1 mol/L/hour, 25 mmol/L/hour to 750mmol/L/hour, 25 mmol/L/hour to 75 mmol/L/hour, 250 mmol/L/hour to 750mmol/L/hour, or 450 mmol/L/hour to 550 mmol/L/hour. In some embodiments,the carbon dioxide evolution rate is about any of 50 mmol/L/hour, 100mmol/L/hour, 150 mmol/L/hour, 200 mmol/L/hour, 250 mmol/L/hour, 300mmol/L/hour, 350 mmol/L/hour, 400 mmol/L/hour, 450 mmol/L/hour, or 500mmol/L/hour.

In addition, provided herein are cells in culture comprising a nucleicacid encoding an isoprene synthase polypeptide, wherein peakconcentration of the isoprene produced by the cells in culture isgreater than about 10 ng/L_(broth) and carbon dioxide evolution rate ofthe cells is greater than about 1×10⁻¹⁸ mmol/L/hour. In someembodiments, the peak concentration of isoprene is any concentration oramount disclosed in the section entitled “Exemplary Production ofIsoprene.” In some embodiments, the peak concentration of isoprene isbetween about any of 10 ng/L_(broth) to 500 ng/L_(broth), 500ng/L_(broth) to 1 μg/L_(broth), 1 μg/L_(broth) to 5 μg/L_(broth), 5μg/L_(broth) to 50 μg/L_(broth), 5 μg/L_(broth) to 100 μg/L_(broth), 5μg/L_(broth) to 250 μg/L_(broth), 250 μg/L_(broth) to 500 μg/L_(broth),500 μg/L_(broth) to 1 mg/L_(broth), 1 mg/L_(broth) to 50 mg/L_(broth), 1mg/L_(broth) to 100 mg/L_(broth), 1 mg/L_(broth) to 200 mg/L_(broth), 10ng/L_(broth) to 200 mg/L_(broth), 5 μg/L_(broth) to 100 mg/L_(broth), or5 μg/L_(broth) to 200 mg/L_(broth). In some embodiments, the peakconcentration is any of about 10 ng/L_(broth), 100 ng/L_(broth), 1μg/L_(broth), 5 μg/L_(broth), 1 mg/L_(broth), 30 mg/L_(broth), 100mg/L_(broth), or 200 mg/L_(broth). In some embodiments, the carbondioxide evolution rate is between about any of 1×10⁻¹⁸ mmol/L/hour toabout 1 mol/L/hour, 1 mmol/L/hour to 1 mol/L/hour, 25 mmol/L/hour to 750mmol/L/hour, 25 mmol/L/hour to 75 mmol/L/hour, 250 mmol/L/hour to 750mmol/L/hour, or 450 mmol/L/hour to 550 mmol/L/hour. In some embodiments,the carbon dioxide evolution rate is about any of 50 mmol/L/hour, 100mmol/L/hour, 150 mmol/L/hour, 200 mmol/L/hour, 250 mmol/L/hour, 300mmol/L/hour, 350 mmol/L/hour, 400 mmol/L/hour, 450 mmol/L/hour, or 500mmol/L/hour.

In some embodiments of any of the methods and cells described herein,carbon dioxide evolution rate and/or cell viability of a cell expressinga MVA pathway and/or DXP pathway RNA and/or protein from one or more ofa heterologous and/or duplicate copy of a MVA pathway and/or DXP pathwaynucleic acid is compared to a control cell lacking one or more of aheterologous and/or duplicate copy of a MVA pathway and/or DXP pathwaynucleic acid. In some embodiments, carbon dioxide evolution rate and/orcell viability of a cell expressing a MVA pathway and/or DXP pathway RNAand/or protein from one or more of a heterologous and/or duplicate copyof a MVA pathway and/or DXP pathway nucleic acid under the control of aninducible promoter, wherein the promotor is induced, is compared to acontrol cell containing one or more of a heterologous and/or duplicatecopy of a MVA pathway and/or DXP pathway nucleic acid under the controlof an inducible promoter, wherein the promotor is not induced(uninduced). In some embodiments, the inducible promoter is abeta-galactosidase promoter.

In some embodiments, the methods of producing isoprene comprise: a)culturing cells under suitable conditions for production of isoprene;and b) producing isoprene, wherein cells produce greater than about 400nmole/g_(wcm)/hour of isoprene, and the carbon dioxide evolution rate ofthe cells is greater than about 1×10⁻¹⁸ mmol/L/hour. Further providedherein are methods of producing isoprene comprising: a) culturing cellsunder suitable conditions for production of isoprene; and b) producingisoprene, wherein the cumulative total productivity of the isopreneproduced by the cells in culture is greater than about 0.2mg/L_(broth)/hour and the carbon dioxide evolution rate of the cells isgreater than about 1×10⁻¹⁸ mmol/L/hour. Methods of producing isopreneare also provided herein comprising: a) culturing cells under suitableconditions for production of isoprene; and b) producing isoprene,wherein the peak concentration of the isoprene produced by the cells inculture is greater than about 10 ng/L_(broth) and the carbon dioxideevolution rate of the cells is greater than about 1×10⁻¹⁸ mmol/L/hour.In some embodiments of any of these methods, the carbon dioxideevolution rate is between about any of 1×10⁻¹⁸ mmol/L/hour to about 1mol/L/hour, 1 mmol/L/hour to 1 mol/L/hour, 25 mmol/L/hour to 750mmol/L/hour, 25 mmol/L/hour to 75 mmol/L/hour, 250 mmol/L/hour to 750mmol/L/hour, or 450 mmol/L/hour to 550 mmol/L/hour. In some embodiments,the carbon dioxide evolution rate is about 50 mmol/L/hour or about 500mmol/L/hour.

Further provided herein are cells in culture comprising a nucleic acidencoding an isoprene synthase polypeptide, wherein the cells producegreater than about 400 nmole/g_(wcm)/hour of isoprene and carbon dioxideevolution rate of the cells is greater than about 1×10⁻¹⁸ mmol/L/hour.Provided herein are also cells in culture comprising a nucleic acidencoding an isoprene synthase polypeptide, wherein cumulative totalproductivity of the isoprene produced by the cells in culture is greaterthan about 0.2 mg/L_(broth)/hour and carbon dioxide evolution rate ofthe cells is greater than about 1×10⁻¹⁸ mmol/L/hour. In addition,provided herein are cells in culture comprising a nucleic acid encodingan isoprene synthase polypeptide, wherein peak concentration of theisoprene produced by the cells in culture is greater than about 10ng/L_(broth) and carbon dioxide evolution rate of the cells is greaterthan about 1×10⁻¹⁸ mmol/L/hour. In some embodiments of any of thesecells in culture, the carbon dioxide evolution rate is between about anyof 1×10⁻¹⁸ mmol/L/hour to about 1 mol/L/hour, 1 mmol/L/hour to 1mol/L/hour, 25 mmol/L/hour to 750 mmol/L/hour, 25 mmol/L/hour to 75mmol/L/hour, 250 mmol/L/hour to 750 mmol/L/hour, or 450 mmol/L/hour to550 mmol/L/hour. In some embodiments, the carbon dioxide evolution rateis about 50 mmol/L/hour or about 500 mmol/L/hour.

Provided herein are also methods of producing isoprene comprising a)culturing cells under suitable conditions for production of isoprene;and b) producing isoprene, wherein the liquid phase concentration ofisoprene is less than about 200 mg/L and the cells produce greater thanabout 400 nmole/g_(wcm)/hour of isoprene. In some embodiments, theliquid phase concentration of isoprene in the culture is less than aboutany of 175 mg/L, 150 mg/L, 125 mg/L, 100 mg/L, 75 mg/L, 50 mg/L, 25mg/L, 20 mg/L, 15 mg/L, 10 mg/L, 5 mg/L, or 2.5 mg/L. In someembodiments, the liquid phase concentration of isoprene in culture isbetween about any of 0.1 mg/L to 200 mg/L, 1 mg/L to 200 mg/L, 1 mg/L to150 mg/L, 1 mg/L to 100 mg/L, 1 mg/L to 50 mg/L, 1 mg/L to 25 mg/L, 1mg/L to 20 mg/L, or 10 mg/L to 20 mg/L.

Also provided herein are methods of producing a compound, wherein thecompound has one or more characteristics selected from the groupconsisting of (a) a Henry's law coefficient of less than about 250 M/atmand (b) a solubility in water of less than about 100 g/L. In someembodiments, the method comprises: a) culturing cells under suitableconditions for production of the compound, wherein gas is added (such asthe addition of gas to a system such as a fermentation system) at a gassparging rate between about 0.01 vvm to about 2 vvm; and b) producingthe compound. In some embodiments, the Henry's law coefficient of thecompound is less than about any of 200 M/atm, 150 M/atm, 100 M/atm, 75M/atm, 50 M/atm, 25 M/atm, 10 M/atm, 5 M/atm, or 1 M/atm. In someembodiments, the solubility in water of the compound is less than aboutany of 75 g/L, 50 g/L, 25 g/L, 10 g/L, 5 g/L, or 1 g/L. In someembodiments, the compound is selected from a group consisting ofisoprene, an aldehyde (e.g., acetaldehyde), a ketone (e.g., acetone or2-butanone), an alcohol (e.g., methanol, ethanol, 1-butanol, or C5alcohols such as 3-methyl-3-buten-1-ol or 3-methyl-2-buten-1-ol), anester of an alcohol (e.g., ethyl acetate or acetyl esters of C5alcohols), a hemiterpene, a monoterpene, a sesquiterpene, and C1 to C5hydrocarbons (e.g., methane, ethane, ethylene, or propylene). In someembodiments, the C1 to C5 hydrocarbons are saturated, unsaturated, orbranched. In particular embodiments, the compound is isoprene. In someembodiments of the methods of producing any of the compounds describedabove, the gas sparging rate is between about any of 0.1 vvm to 1 vvm,0.2 vvm to 1 vvm, or 0.5 vvm to 1 vvm.

In one aspect, cells in culture are used to produce isoprene. In someembodiments, the cells in culture produce greater than about 400 nmoleof isoprene/gram of cells for the wet weight of the cells/hour(nmole/g_(wcm)/hr) of isoprene. In some embodiments, the cells have aheterologous nucleic acid that (i) encodes an isoprene synthasepolypeptide and (ii) is operably linked to a promoter. In someembodiments, the cells are cultured in a culture medium that includes acarbon source, such as, but not limited to, a carbohydrate, glycerol,glycerine, dihydroxyacetone, one-carbon source, oil, animal fat, animaloil, fatty acid, lipid, phospholipid, glycerolipid, monoglyceride,diglyceride, triglyceride, renewable carbon source, polypeptide (e.g., amicrobial or plant protein or peptide), yeast extract, component from ayeast extract, or any combination of two or more of the foregoing. Insome embodiments, the cells are cultured under limited glucoseconditions.

In some embodiments, the cells in culture convert more than about 0.002%of the carbon in a cell culture medium into isoprene. In someembodiments, the cells have a heterologous nucleic acid that (i) encodesan isoprene synthase polypeptide and (ii) is operably linked to apromoter. In some embodiments, the cells are cultured in a culturemedium that includes a carbon source, such as, but not limited to, acarbohydrate, glycerol, glycerine, dihydroxyacetone, one-carbon source,oil, animal fat, animal oil, fatty acid, lipid, phospholipid,glycerolipid, monoglyceride, diglyceride, triglyceride, renewable carbonsource, polypeptide (e.g., a microbial or plant protein or peptide),yeast extract, component from a yeast extract, or any combination of twoor more of the foregoing. In some embodiments, the cells are culturedunder limited glucose conditions.

In some embodiments, the cells in culture comprise a heterologousnucleic acid encoding an isoprene synthase polypeptide. In someembodiments, the cells have a heterologous nucleic acid that (i) encodesan isoprene synthase polypeptide and (ii) is operably linked to apromoter. In some embodiments, the cells are cultured in a culturemedium that includes a carbon source, such as, but not limited to, acarbohydrate, glycerol, glycerine, dihydroxyacetone, one-carbon source,oil, animal fat, animal oil, fatty acid, lipid, phospholipid,glycerolipid, monoglyceride, diglyceride, triglyceride, renewable carbonsource, polypeptide (e.g., a microbial or plant protein or peptide),yeast extract, component from a yeast extract, or any combination of twoor more of the foregoing. In some embodiments, the cells are culturedunder limited glucose conditions.

In one aspect, described herein are methods of producing isoprene, suchas methods of using any of the cells described herein to produceisoprene. In some embodiments, the method involves culturing cells underconditions sufficient to produce greater than about 400 nmole/g_(wcm)/hrof isoprene. In some embodiments, the method also includes recoveringisoprene produced by the cells. In some embodiments, the method includespurifying isoprene produced by the cells. In some embodiments, themethod includes polymerizing the isoprene. In some embodiments, thecells have a heterologous nucleic acid that (i) encodes an isoprenesynthase polypeptide and (ii) is operably linked to a promoter. In someembodiments, the cells are cultured in a culture medium that includes acarbon source, such as, but not limited to, a carbohydrate, glycerol,glycerine, dihydroxyacetone, one-carbon source, oil, animal fat, animaloil, fatty acid, lipid, phospholipid, glycerolipid, monoglyceride,diglyceride, triglyceride, renewable carbon source, polypeptide (e.g., amicrobial or plant protein or peptide), yeast extract, component from ayeast extract, or any combination of two or more of the foregoing. Insome embodiments, the cells are cultured under limited glucoseconditions. In various embodiments, the amount of isoprene produced(such as the total amount of isoprene produced or the amount of isopreneproduced per liter of broth per hour per OD₆₀₀) during stationary phaseis greater than or about 2 or more times the amount of isoprene producedduring the growth phase for the same length of time. In someembodiments, the gas phase comprises greater than or about 9.5% (volume)oxygen, and the concentration of isoprene in the gas phase is less thanthe lower flammability limit or greater than the upper flammabilitylimit. In particular embodiments, (i) the concentration of isoprene inthe gas phase is less than the lower flammability limit or greater thanthe upper flammability limit, and (ii) the cells produce greater thanabout 400 nmole/g_(wcm)/hr of isoprene.

In some embodiments, the method includes culturing cells underconditions sufficient to convert more than about 0.002% of the carbon(mol/mol) in a cell culture medium into isoprene. In some embodiments,the method also includes recovering isoprene produced by the cells. Insome embodiments, the method includes purifying isoprene produced by thecells. In some embodiments, the method includes polymerizing theisoprene. In some embodiments, the cells have a heterologous nucleicacid that (i) encodes an isoprene synthase polypeptide and (ii) isoperably linked to a promoter. In some embodiments, the cells arecultured in a culture medium that includes a carbon source, such as, butnot limited to, a carbohydrate, glycerol, glycerine, dihydroxyacetone,one-carbon source, oil, animal fat, animal oil, fatty acid, lipid,phospholipid, glycerolipid, monoglyceride, diglyceride, triglyceride,renewable carbon source, polypeptide (e.g., a microbial or plant proteinor peptide), yeast extract, component from a yeast extract, or anycombination of two or more of the foregoing. In some embodiments, thecells are cultured under limited glucose conditions.

In some embodiments, isoprene is only produced in stationary phase. Insome embodiments, isoprene is produced in both the growth phase andstationary phase. In various embodiments, the amount of isopreneproduced (such as the total amount of isoprene produced or the amount ofisoprene produced per liter of broth per hour per OD₆₀₀) duringstationary phase is greater than or about 2, 3, 4, 5, 10, 20, 30, 40,50, or more times the amount of isoprene produced during the growthphase for the same length of time.

In one aspect, described herein are compositions and systems thatcomprise isoprene. In some embodiments, the composition comprisesgreater than or about 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,200, 300, 400, 500, 600, 700, 800, 900, or 1000 mg of isoprene. In someembodiments, the composition comprises greater than or about 2, 5, 10,20, 30, 40, 50, 60, 70, 80, 90, 100 g of isoprene(w/w) of the volatileorganic fraction of the composition is isoprene.

In some embodiments, the composition comprises greater than or about99.90, 99.92, 99.94, 99.96, 99.98, or 100% isoprene by weight comparedto the total weight of all C5 hydrocarbons in the composition. In someembodiments, the composition comprises less than or about 0.12, 0.10,0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or0.00001% C5 hydrocarbons other than isoprene (such as1,3-cyclopentadiene, cis-1,3-pentadiene, trans-1,3-pentadiene,1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene, 2-methyl-1-butene,3-methyl-1-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, orcis-pent-3-ene-1-yne) by weight compared to the total weight of all C5hydrocarbons in the composition. In some embodiments, the compositionhas less than or about 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005,0.001, 0.0005, 0.0001, 0.00005, or 0.00001% for 1,3-cyclopentadiene,cis-1,3-pentadiene, trans-1,3-pentadiene, 1,4-pentadiene, 1-pentyne,2-pentyne, 1-pentene, 2-methyl-1-butene, 3-methyl-1-butyne,pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne byweight compared to the total weight of all C5 hydrocarbons in thecomposition. In particular embodiments, the composition has greater thanabout 2 mg of isoprene and has greater than or about 99.90, 99.92,99.94, 99.96, 99.98, or 100% isoprene by weight compared to the totalweight of all C5 hydrocarbons in the composition.

In some embodiments, the composition has less than or about 50, 40, 30,20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 μg/L of a compound thatinhibits the polymerization of isoprene for any compound in thecomposition that inhibits the polymerization of isoprene. In particularembodiments, the composition also has greater than about 2 mg ofisoprene.

In some embodiments, the composition has one or more compounds selectedfrom the group consisting of ethanol, acetone, C5 prenyl alcohols, andisoprenoid compounds with 10 or more carbon atoms. In some embodiments,the composition has greater than or about 0.005, 0.01, 0.05, 0.1, 0.5,1, 5, 10, 20, 30, 40, 60, 80, 100, or 120 μg/L of ethanol, acetone, a C5prenyl alcohol (such as 3-methyl-3-buten-1-ol or 3-methyl-2-buten-1-ol),or any two or more of the foregoing. In particular embodiments, thecomposition has greater than about 2 mg of isoprene and has one or morecompounds selected from the group consisting of ethanol, acetone, C5prenyl alcohols, and isoprenoid compounds with 10 or more carbon atoms.

In some embodiments, the composition includes isoprene and one or moresecond compounds selected from the group consisting of 2-heptanone,6-methyl-5-hepten-2-one, 2,4,5-trimethylpyridine,2,3,5-trimethylpyrazine, citronellal, acetaldehyde, methanethiol, methylacetate, 1-propanol, diacetyl, 2-butanone, 2-methyl-3-buten-2-ol, ethylacetate, 2-methyl-1-propanol, 3-methyl-1-butanal, 3-methyl-2-butanone,1-butanol, 2-pentanone, 3-methyl-1-butanol, ethyl isobutyrate,3-methyl-2-butenal, butyl acetate, 3-methylbutyl acetate,3-methyl-3-buten-1-yl acetate, 3-methyl-2-buten-1-yl acetate,3-hexen-1-ol, 3-hexen-1-yl acetate, limonene, geraniol(trans-3,7-dimethyl-2,6-octadien-1-ol), citronellol(3,7-dimethyl-6-octen-1-ol), (E)-3,7-dimethyl-1,3,6-octatriene,(Z)-3,7-dimethyl-1,3,6-octatriene, and 2,3-cycloheptenolpyridine. Invarious embodiments, the amount of one of these second componentsrelative to the amount of isoprene in units of percentage by weight(i.e., weight of the component divided by the weight of isoprene times100) is at greater than or about 0.01, 0.02, 0.05, 0.1, 0.5, 1, 5, 10,20, 30, 40, 50, 60, 70, 80, 90, 100, or 110% (w/w).

In some embodiments, the composition comprises (i) a gas phase thatcomprises isoprene and (ii) cells in culture that produce greater thanabout 400 nmole/g_(wcm)/hr of isoprene. In some embodiments, thecomposition comprises a closed system, and the gas phase comprisesgreater than or about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 μg/L ofisoprene when normalized to 1 mL of 1 OD₆₀₀ cultured for 1 hour. In someembodiments, the composition comprises an open system, and the gas phasecomprises greater than or about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90,100 μg/L of isoprene when sparged at a rate of 1 vvm. In someembodiments, the volatile organic fraction of the gas phase comprisesgreater than or about 99.90, 99.92, 99.94, 99.96, 99.98, or 100%isoprene by weight compared to the total weight of all C5 hydrocarbonsin the volatile organic fraction. In some embodiments, the volatileorganic fraction of the gas phase comprises less than or about 0.12,0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001,0.00005, or 0.00001% C5 hydrocarbons other than isoprene (such as1,3-cyclopentadiene, cis-1,3-pentadiene, trans-1,3-pentadiene,1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene, 2-methyl-1-butene,3-methyl-1-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, orcis-pent-3-ene-1-yne) by weight compared to the total weight of all C5hydrocarbons in the volatile organic fraction. In some embodiments, thevolatile organic fraction of the gas phase has less than or about 0.12,0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001,0.00005, or 0.00001% for 1,3-cyclopentadiene, cis-1,3-pentadiene,trans-1,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene,2-methyl-1-butene, 3-methyl-1-butyne, pent-4-ene-1-yne,trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne by weight compared tothe total weight of all C5 hydrocarbons in the volatile organicfraction. In particular embodiments, the volatile organic fraction ofthe gas phase has greater than about 2 mg of isoprene and has greaterthan or about 99.90, 99.92, 99.94, 99.96, 99.98, or 100% isoprene byweight compared to the total weight of all C5 hydrocarbons in thevolatile organic fraction.

In some embodiments, the volatile organic fraction of the gas phase hasless than or about 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or0.005 μg/L of a compound that inhibits the polymerization of isoprenefor any compound in the volatile organic fraction of the gas phase thatinhibits the polymerization of isoprene. In particular embodiments, thevolatile organic fraction of the gas phase also has greater than about 2mg of isoprene.

In some embodiments, the volatile organic fraction of the gas phase hasone or more compounds selected from the group consisting of ethanol,acetone, C5 prenyl alcohols, and isoprenoid compounds with 10 or morecarbon atoms. In some embodiments, the volatile organic fraction of thegas phase has greater than or about 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5,10, 20, 30, 40, 60, 80, 100, or 120 μg/L of ethanol, acetone, a C5prenyl alcohol (such as 3-methyl-3-buten-1-ol or 3-methyl-2-buten-1-ol),or any two or more of the foregoing. In particular embodiments, thevolatile organic fraction of the gas phase has greater than about 2 mgof isoprene and has one or more compounds selected from the groupconsisting of ethanol, acetone, C5 prenyl alcohols, and isoprenoidcompounds with 10 or more carbon atoms.

In some embodiments, the volatile organic fraction of the gas phase hasincludes isoprene and one or more second compounds selected from thegroup consisting of 2-heptanone, 6-methyl-5-hepten-2-one,2,4,5-trimethylpyridine, 2,3,5-trimethylpyrazine, citronellal,acetaldehyde, methanethiol, methyl acetate, 1-propanol, diacetyl,2-butanone, 2-methyl-3-buten-2-ol, ethyl acetate, 2-methyl-1-propanol,3-methyl-1-butanal, 3-methyl-2-butanone, 1-butanol, 2-pentanone,3-methyl-1-butanol, ethyl isobutyrate, 3-methyl-2-butenal, butylacetate, 3-methylbutyl acetate, 3-methyl-3-buten-1-yl acetate,3-methyl-2-buten-1-yl acetate, 3-hexen-1-ol, 3-hexen-1-yl acetate,limonene, geraniol (trans-3,7-dimethyl-2,6-octadien-1-ol), citronellol(3,7-dimethyl-6-octen-1-ol), (E)-3,7-dimethyl-1,3,6-octatriene,(Z)-3,7-dimethyl-1,3,6-octatriene, and 2,3-cycloheptenolpyridine. Invarious embodiments, the amount of one of these second componentsrelative to amount of isoprene in units of percentage by weight (i.e.,weight of the component divided by the weight of isoprene times 100) isat greater than or about 0.01, 0.02, 0.05, 0.1, 0.5, 1, 5, 10, 20, 30,40, 50, 60, 70, 80, 90, 100, or 110% (w/w) in the volatile organicfraction of the gas phase.

In some embodiments of any of the compositions described herein, atleast a portion of the isoprene is in a gas phase. In some embodiments,at least a portion of the isoprene is in a liquid phase (such as acondensate). In some embodiments, at least a portion of the isoprene isin a solid phase. In some embodiments, at least a portion of theisoprene is adsorbed to a solid support, such as a support that includessilica and/or activated carbon. In some embodiments, the compositionincludes ethanol. In some embodiments, the composition includes betweenabout 75 to about 90% by weight of ethanol, such as between about 75 toabout 80%, about 80 to about 85%, or about 85 to about 90% by weight ofethanol. In some embodiments, the composition includes between about 4to about 15% by weight of isoprene, such as between about 4 to about 8%,about 8 to about 12%, or about 12 to about 15% by weight of isoprene.

In some embodiments, the systems include any of the cells and/orcompositions described herein. In some embodiments, the system includesa reactor that chamber comprises cells in culture that produce greaterthan about 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750,2,000, 2,500, 3,000, 4,000, 5,000, or more nmole/g_(wcm)/hr isoprene. Insome embodiments, the system is not a closed system. In someembodiments, at least a portion of the isoprene is removed from thesystem. In some embodiments, the system includes a gas phase comprisingisoprene. In various embodiments, the gas phase comprises any of thecompositions described herein.

In some embodiments of any of the compositions, systems, and methodsdescribed herein, a nonflammable concentration of isoprene in the gasphase is produced. In some embodiments, the gas phase comprises lessthan about 9.5% (volume) oxygen. In some embodiments, the gas phasecomprises greater than or about 9.5% (volume) oxygen, and theconcentration of isoprene in the gas phase is less than the lowerflammability limit or greater than the upper flammability limit. In someembodiments, the portion of the gas phase other than isoprene comprisesbetween about 0% to about 100% (volume) oxygen, such as between about10% to about 100% (volume) oxygen. In some embodiments, the portion ofthe gas phase other than isoprene comprises between about 0% to about99% (volume) nitrogen. In some embodiments, the portion of the gas phaseother than isoprene comprises between about 1% to about 50% (volume)CO₂.

In some embodiments of any of the aspects described herein, the cells inculture produce isoprene at greater than or about 400, 500, 600, 700,800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000,or more nmole/g_(wcm)/hr isoprene. In some embodiments of any of theaspects described herein, the cells in culture convert greater than orabout 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.12, 0.14, 0.16, 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6%, or more of the carbonin the cell culture medium into isoprene. In some embodiments of any ofthe aspects described herein, the cells in culture produce isoprene atgreater than or about 1, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500,600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000,4,000, 5,000, 10,000, 100,000, or more ng of isoprene/gram of cells forthe wet weight of the cells/hr (ng/g_(wcm)/h). In some embodiments ofany of the aspects described herein, the cells in culture produce acumulative titer (total amount) of isoprene at greater than or about 1,10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900,1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000, 10,000,50,000, 100,000, or more mg of isoprene/L of broth (mg/L_(broth),wherein the volume of broth includes the volume of the cells and thecell medium). Other exemplary rates of isoprene production and totalamounts of isoprene production are disclosed herein.

In some embodiments of any of the aspects described herein, the cellsfurther comprise a heterologous nucleic acid encoding an IDIpolypeptide. In some embodiments of any of the aspects described herein,the cells further comprise an insertion of a copy of an endogenousnucleic acid encoding an IDI polypeptide. In some embodiments of any ofthe aspects described herein, the cells further comprise a heterologousnucleic acid encoding a DXS polypeptide. In some embodiments of any ofthe aspects described herein, the cells further comprise an insertion ofa copy of an endogenous nucleic acid encoding a DXS polypeptide. In someembodiments of any of the aspects described herein, the cells furthercomprise one or more nucleic acids encoding an IDI polypeptide and a DXSpolypeptide. In some embodiments of any of the aspects described herein,one nucleic acid encodes the isoprene synthase polypeptide, IDIpolypeptide, and DXS polypeptide. In some embodiments of any of theaspects described herein, one vector encodes the isoprene synthasepolypeptide, IDI polypeptide, and DXS polypeptide. In some embodiments,the vector comprises a selective marker, such as an antibioticresistance nucleic acid.

In some embodiments of any of the aspects described herein, theheterologous isoprene synthase nucleic acid is operably linked to a T7promoter, such as a T7 promoter contained in a medium or high copyplasmid. In some embodiments of any of the aspects described herein, theheterologous isoprene synthase nucleic acid is operably linked to a Trcpromoter, such as a Trc promoter contained in a medium or high copyplasmid. In some embodiments of any of the aspects described herein, theheterologous isoprene synthase nucleic acid is operably linked to a Lacpromoter, such as a Lac promoter contained in a low copy plasmid. Insome embodiments of any of the aspects described herein, theheterologous isoprene synthase nucleic acid is operably linked to anendogenous promoter, such as an endogenous alkaline serine proteasepromoter. In some embodiments, the heterologous isoprene synthasenucleic acid integrates into a chromosome of the cells without aselective marker.

In some embodiments, one or more MVA pathway, IDI, DXP, or isoprenesynthase nucleic acids are placed under the control of a promoter orfactor that is more active in stationary phase than in the growth phase.For example, one or more MVA pathway, IDI, DXP, or isoprene synthasenucleic acids may be placed under control of a stationary phase sigmafactor, such as RpoS. In some embodiments, one or more MVA pathway, IDI,DXP, or isoprene synthase nucleic acids are placed under control of apromoter inducible in stationary phase, such as a promoter inducible bya response regulator active in stationary phase.

In some embodiments of any of the aspects described herein, at least aportion of the cells maintain the heterologous isoprene synthase nucleicacid for at least or about 5, 10, 20, 40, 50, 60, 65, or more celldivisions in a continuous culture (such as a continuous culture withoutdilution). In some embodiments of any of the aspects described herein,the nucleic acid comprising the isoprene synthase, IDI, or DXS nucleicacid also comprises a selective marker, such as an antibiotic resistancenucleic acid.

In some embodiments of any of the aspects described herein, the cellsfurther comprise a heterologous nucleic acid encoding an MVA pathwaypolypeptide (such as an MVA pathway polypeptide from Saccharomycescerevisia or Enterococcus faecalis). In some embodiments of any of theaspects described herein, the cells further comprise an insertion of acopy of an endogenous nucleic acid encoding an MVA pathway polypeptide(such as an MVA pathway polypeptide from Saccharomyces cerevisia orEnterococcus faecalis). In some embodiments of any of the aspectsdescribed herein, the cells comprise an isoprene synthase, DXS, and MVApathway nucleic acid. In some embodiments of any of the aspectsdescribed herein, the cells comprise an isoprene synthase nucleic acid,a DXS nucleic acid, an IDI nucleic acid, and a MVA pathway nucleic (inaddition to the IDI nucleic acid).

In some embodiments of any of the aspects described herein, the isoprenesynthase polypeptide is a polypeptide from a plant such as Pueraria(e.g., Pueraria montana or Pueraria lobata) or Populus (e.g., Populustremuloides, Populus alba, Populus nigra, Populus trichocarpa, or thehybrid, Populus alba×Populus tremula).

In some embodiments of any of the aspects described herein, the cellsare bacterial cells, such as gram-positive bacterial cells (e.g.,Bacillus cells such as Bacillus subtilis cells or Streptomyces cellssuch as Streptomyces lividans, Streptomyces coelicolor, or Streptomycesgriseus cells). In some embodiments of any of the aspects describedherein, the cells are gram-negative bacterial cells (e.g., Escherichiacells such as Escherichia coli cells, Rhodopseudomonas sp. such asRhodopseudomonas palustris cells, Pseudomonas sp. such as Pseudomonasfluorescens cells or Pseudomonas putida cells, or Pantoea cells such asPantoea citrea cells). In some embodiments of any of the aspectsdescribed herein, the cells are fungal, cells such as filamentous fungalcells (e.g., Trichoderma cells such as Trichoderma reesei cells orAspergillus cells such as Aspergillus oryzae and Aspergillus niger) oryeast cells (e.g., Yarrowia cells such as Yarrowia lipolytica cells orSacchraomyces cells such as Saccaromyces cerevisiae).

In some embodiments of any of the aspects described herein, themicrobial polypeptide carbon source includes one or more polypeptidesfrom yeast or bacteria. In some embodiments of any of the aspectsdescribed herein, the plant polypeptide carbon source includes one ormore polypeptides from soy, corn, canola, jatropha, palm, peanut,sunflower, coconut, mustard, rapeseed, cottonseed, palm kernel, olive,safflower, sesame, or linseed.

Production of Isoprene in Anaerobic Microorganisms Using Synthesis Gasas an Energy Source

In some embodiments, the bioisoprene composition is produced inanaerobic microorganisms using synthesis gas as an energy source asdescribed in U.S. Provisional Patent Application Nos. 61/289,347 and61/289,355, filed on Dec. 22, 2009, the disclosures of which areincorporated herein by reference in their entireties.

Production of isoprene from syngas by anaerobic organisms may provide anumber of advantages over production of isoprene from sugars by aerobicorganisms. First, the maximum theoretical mass yield of isoprene can begreater for the aerobic organisms, as discussed further below. Second,the anaerobic organisms do not have excess reducing power in the form ofNAD(P)H that must be turned over via cell growth, formation ofbyproducts (such as glycerol, lactic acid, or ethanol) or oxidationusing molecular oxygen. Without this NAD(P)H turnover requirement,anaerobic organisms can have higher energy yield, lower oxygen demand,lower heat of fermentation, and lower utility costs to run the process.Third, due to the lack of oxygen in the system, anaerobic organisms canhave greater isoprene concentration in the offgas, lower probability ofcreating a flammable isoprene-oxygen mixture, easier recovery, andhigher isoprene quality. Fourth, the anaerobic organisms can be moreeasily grown by using existing infrastructure, such as existing plantsdesigned for production of bioethanol.

Anaerobic organisms useful for isoprene production can include obligateanaerobes, facultatitive anaerobes, and aerotolerant anaerobes. Theobligate anaerobes can be any one or combination selected from the groupconsisting of Clostridium ljungdahlii, Clostridium autoethanogenum,Eurobacterium limosum, Clostridium carboxydivorans, Peptostreptococcusproductus, and Butyribacterium methylotrophicum. Syngas useful as energysource for production of isoprene can be derived from a feedstock by avariety of processes, including methane reforming, coal liquefaction,co-firing, fermentative reactions, enzymatic reactions, and biomassgasification.

Exemplary Purification Methods

In some embodiments, any of the methods described herein further includerecovering the co-produced compounds. In some embodiments, any of themethods described herein further include recovering the isoprene. Insome embodiments, any of the methods described herein further includerecovering the hydrogen by cryogenic membrane, adsorption matrix-basedseparation methods.

The isoprene and hydrogen produced using the compositions and methodsdescribed herein can be recovered using standard techniques. such as gasstripping, membrane enhanced separation, fractionation,adsorption/desorption, pervaporation, thermal or vacuum desorption ofisoprene from a solid phase, or extraction of isoprene immobilized orabsorbed to a solid phase with a solvent (see, for example, U.S. Pat.Nos. 4,703,007, 4,570,029, and 4,740,222 (“Recovery and Purification ofHydrogen from Refinery and Petrochemical Off-gas Streams”) which areeach hereby incorporated by reference in their entireties, particularlywith respect to isoprene recovery and purification methods ('007 and'029 patents) and with respect to hydrogen recovery and purificationmethods ('222 patent)). In particular embodiments, extractivedistillation with an alcohol (such as ethanol, methanol, propanol, or acombination thereof) is used to recover the isoprene. In someembodiments, the recovery of isoprene involves the isolation of isoprenein a liquid form (such as a neat solution of isoprene or a solution ofisoprene in a solvent). Gas stripping involves the removal of isoprenevapor from the fermentation off-gas stream in a continuous manner. Suchremoval can be achieved in several different ways including, but notlimited to, adsorption to a solid phase, partition into a liquid phase,or direct condensation (such as condensation due to exposure to acondensation coil or do to an increase in pressure). In someembodiments, membrane enrichment of a dilute isoprene vapor stream abovethe dew point of the vapor resulting in the condensation of liquidisoprene. In some embodiments, the isoprene is compressed and condensed.

The recovery of isoprene may involve one step or multiple steps. In someembodiments, the removal of isoprene vapor from the fermentation off-gasand the conversion of isoprene to a liquid phase are performedsimultaneously. For example, isoprene can be directly condensed from theoff-gas stream to form a liquid. In some embodiments, the removal ofisoprene vapor from the fermentation off-gas and the conversion ofisoprene to a liquid phase are performed sequentially. For example,isoprene may be adsorbed to a solid phase and then extracted from thesolid phase with a solvent.

The recovery of hydrogen may involve one step or multiple steps. In someembodiments, the removal of hydrogen gas from the fermentation off-gasand the conversion of hydrogen to a liquid phase are performedsimultaneously. In some embodiments, the removal of hydrogen gas fromthe fermentation off-gas and the conversion of hydrogen to a liquidphase are performed sequentially. For example, hydrogen may be adsorbedto a solid phase and then desorbed from the solid phase by a pressureswing. In some embodiments, recovered hydrogen gas is concentrated andcompressed.

In some embodiments, any of the methods described herein further includepurifying the isoprene. For example, the isoprene produced using thecompositions and methods described herein can be purified using standardtechniques. Purification refers to a process through which isoprene isseparated from one or more components that are present when the isopreneis produced. In some embodiments, the isoprene is obtained as asubstantially pure liquid. Examples of purification methods include (i)distillation from a solution in a liquid extractant and (ii)chromatography. As used herein, “purified isoprene” means isoprene thathas been separated from one or more components that are present when theisoprene is produced. In some embodiments, the isoprene is at leastabout 20%, by weight, free from other components that are present whenthe isoprene is produced. In various embodiments, the isoprene is atleast or about 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, or 99%,by weight, pure. Purity can be assayed by any appropriate method, e.g.,by column chromatography, HPLC analysis, or GC-MS analysis.

In some embodiments, any of the methods described herein further includepurifying the hydrogen. For example, the hydrogen produced using thecompositions and methods described herein can be purified using standardtechniques. Purification refers to a process through which hydrogen isseparated from one or more components that are present when the hydrogenis produced. In some embodiments, the hydrogen is obtained as asubstantially pure gas. In some embodiments, the hydrogen is obtained asa substantially pure liquid. Examples of purification methods include(i) cryogenic condensation and (ii) solid matrix adsorption. As usedherein, “purified hydrogen” means hydrogen that has been separated fromone or more components that are present when the hydrogen is produced.In some embodiments, the hydrogen is at least about 20%, by weight, freefrom other components that are present when the hydrogen is produced. Invarious embodiments, the hydrogen is at least or about 25%, 30%, 40%,50%, 60%, 70%, 75%, 80%, 90%, 95%, or 99%, by weight, pure. Purity canbe assayed by any appropriate method, e.g., by column chromatography orGC-MS analysis.

In some embodiments, at least a portion of the gas phase remaining afterone or more recovery steps for the removal of isoprene is recycled byintroducing the gas phase into a cell culture system (such as afermentor) for the production of isoprene.

Methods and apparatus for the purification of a bioisoprene compositionfrom fermentor off-gas is described in U.S. Provisional PatentApplication No. 61/88,142, filed Dec. 18, 2009, which is incorporatedherein by reference in its entirety.

A bioisoprene composition from a fermentor off-gas may containbioisoprene with volatile impurities and bio-byproduct impurities. Insome embodiments, a bioisoprene composition from a fermentor off-gas ispurified using a method comprising: (a) contacting the fermentor off-gaswith a solvent in a first column to form: an isoprene-rich solutioncomprising the solvent, a major portion of the isoprene and a majorportion of the bio-byproduct impurity; and a vapor comprising a majorportion of the volatile impurity; (b) transferring the isoprene-richsolution from the first column to a second column; and (c) strippingisoprene from the isoprene-rich solution in the second column to form:an isoprene-lean solution comprising a major portion of thebio-byproduct impurity; and a purified isopene composition.

FIG. 169 illustrates an exemplary method of purifying isoprene and anexemplary apparatus. Fermentor off-gas comprising isoprene may begenerated from renewable resources (e.g., carbon sources) by any methodin the art for example, as described in U.S. provisional patentapplication Nos. 61/187,944, the content of which is hereby incorporatedby reference, particularly with respect to the methods of generatingfermentor off-gas comprising isoprene. The fermentor off-gas generatedfrom one or more individual fermentors 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8,or more fermentors connected in series and/or in parallel) may bedirected to a first column 14. As described below, the fermentor off-gasmay be directed through an isolation unit 16 and/or compressed by acompression means, such as compression system 18. Additionally, thetemperature of the fermentor off-gas may optionally be reduced at anypoint, for example, to form a condensate or partial condensate prior tocontact with the solvent (which may aid in solubiliztion of one or moreoff-gas components, such as isoprene). The fermentor off-gas may becontacted (e.g., absorbed) at column 14 with a solvent (e.g., anysolvent described herein, such as a non-polar high boiling-pointsolvent). The volatile impurities having less propensity for absorptionin the solvent (particularly with non-polar high boiling-point solvents)are separated from the remaining solvent/fermentor off-gas mixture,resulting in a vapor comprising a major portion of the volatile impurity(e.g., exiting at port 20), and an isoprene-rich solution having a majorportion of the isoprene and a major portion of the bio-byproductimpurity (e.g., at port 22). The solvent may optionally be heated by anysuitable means (e.g., by steam) prior to, simultaneously, and/or aftercontact with the fermentor off-gas, which may aid in separation of thevolatile impurity from the remaining solution. Steam may be directedthrough the column (at any suitable location, such as near entry of theoff-gas and/or the opposite end of the volatile impurity exit as shownin FIG. 169) to provide a sweeping vapor phase which may aid in theremoval of the volatile impurity.

The isoprene-rich solution having a major portion of the isoprene and amajor portion of the bio-byproduct impurity (e.g., at port 22) may bedirected to a second column 24. The second column may be isolated fromthe first column 14 (as shown in FIG. 169) or may be part of a singlecolumn comprising both the first and second columns (e.g., a tandemcolumn wherein the solvent enters the first column at or near one end,and exits the second column at or near an opposite end). The isoprenemay be stripped from the isoprene-rich solution in the second column togenerate a purified isopene composition (e.g., at port 26) and anisoprene-lean solution comprising a major portion of the bio-byproductimpurity (e.g., at port 28). The isoprene-rich solution may be heated byany suitable means (e.g., by steam), which may aid in stripping of theisoprene from the remaining solution. Steam may be directed through thecolumn (at any suitable location, such as the opposite end of the entrypoint of the isoprene-rich solution and/or the near the end of theisoprene-lean solution exit as shown in FIG. 169).

As described herein, the columns may be conventional and of any suitablesize. Exemplary types of columns are commercially available frommanufacturers including Koch Modular Process Systems (Paramus, N.J.),Fluor Corporation (Irving, Tex.), Kuhni USA (Mount Holly, N.C.). Ingeneral, columns are designed to maximize vapor/liquid contact in orderto achieve the desired efficiency. This is achieved by filling thecolumn with either a packing material, or trays spaced at regularintervals along the column. Suitable packing materials include bothrandom and structured types based on metal, glass, polymer and ceramicmaterials. Exemplary random packing types include Raschig rings, Pallrings, A-PAK rings, Saddle rings, Pro-Pak, Heli-Pak, Ceramic saddles andFLEXIRINGS®. Structured packings include wire mesh and perforated metalplate type materials. Manfacturers specializing in column packingsinclude ACS Separations & Mass-Transfer Products (Houston, Tex.),Johnson Bros. Metal Forming Co. (Berkeley, Ill.) and Koch Glitsch, Inc.Knight Div. (East Canton, Ohio). The efficiency of a gas strippingcolumn is expressed in terms of the theoretical plate height and thetotal number of plates in the column. In general, the greater the numberof theoretical plates present, the greater the efficiency of the column.Laboratory scale columns can be purchased from Ace Glass (Vineland,N.J.), Sigma-Aldrich (St. Louis, Mo.) and Chemglass (Vineland, N.J.).Suitable types of glass column include Vigreux, Snyder, Hemple andPerforated-plate type columns. Columns can include packing materals, orcontain features designed to maximize vapor/liquid contact. A laboratoryscale gas scrubber unit (part # CG-1830-10) is available from Chemglassand consists of a packed glass column, solvent reservoir and solventrecirculation pump.

The purified isoprene composition from the second column 24 (e.g.,exiting at port 26) may be further purified by any suitable means (e.g.,by using a reflux condenser 34 and/or an adsorption system 36, such as asilica adsorption system). The reflux reduces the solvent composition inthe isoprene product. The isoprene-lean solution may be recycled back tothe first column for reuse (e.g., as shown in FIG. 169 at port 30). Theisoprene-lean solution may be purified by any suitable means (e.g., byliquid-liquid extraction and/or an adsorption system 32, such as asilica adsorption system) prior to recycling to the first column 14 toreduce to amount of bio-byproduct. Additionally, the temperature of theisoprene-lean solution may be reduced by any suitable means prior torecycling to the first column 14 (e.g., prior to, simultaneously, and/orafter optionally purifying the isoprene solution). FIG. 169 shows anexample of reducing the temperature of the isoprene-lean solution atport 40 prior to purification of the isoprene-lean solution (in thiscase, using coolant for temperature reduction).

The vapor comprising a major portion of the volatile impurity (e.g., thevapor exiting at port 20 in FIG. 169) may comprise a minor portion ofisoprene (e.g., residual isoprene not remaining in the isoprene-richsolution). The residual isoprene may be recollected for use from thevapor comprising a major portion of the volatile impurity by anysuitable means (e.g., an adsorption system 38, such as an activatedcarbon adsorption system) and in some cases, as shown in FIG. 169, maybe combined with the purified isoprene composition (e.g., prior to,during, or after additional purification, such as an adsorption systemsimilar to system 36). FIG. 169 also shows an optional capture device 42(e.g., a thermal oxidizer and/or CO₂ capture system) capable of reducingthe amount of undesirable components released into the atmosphere (e.g.,CO₂) from the vapor.

Exemplary Chemical Transformations of Isoprene

Although current industrial use of isoprene is predominantly in theproduction of synthetic rubber, isoprene is a reactive conjugated dieneand undergoes a varieties of chemical transformations to form oxygenatesand higher molecular weight hydrocarbons. For example, Palladium(0)complexes (Pd(acac)₂-Ph₃P and Pd(OAc)₂-Ph₃P) catalyze dimerization andtelomerization of isoprene in alcohol solvents to give linear isoprenedimers (e.g. 2,7-dimethyl-1,3,7-octatriene) andmethoxydimethyloctadienes (Zakharkin, L. I. and Babich, S. A. Russ.Chem. Bull. (1976), pp 1967-1968.) Adams, J. M. and Clapp, T. V. (Clayand Clay Minerals (1986), 34(3), 287-294) reported reactions of isopreneover divalent and trivalent transition metal-exchanged montmorillonites(e.g. Cr³⁺-montmorillonite) to give isoprene dimers and adducts withmethanol. The linear dimerization of isoprene catalyzed byNi(0)-aminophosphinite systems resulted in regioselective tail-to-taillinear dimers, accompanied by a competitive cyclodimerization reaction(Denis, Philippe; Croizy, Jean Francois; Mortreux, Andre; Petit,Francis, Journal of Molecular Catalysis (1991), 68(2), 159-75. Denis,Philippe; Jean, Andre; Croizy, Jean Francois; Mortreux, Andre; Petit,Francis, Journal of the American Chemical Society (1990), 112(3),1292-4.) New chiral aminophosphinite ligands, e.g.,(+)-MeCH₂CHMeCH(NH₂)CH₂OPPh₂ was investigated as homogeneous catalystsin the linear dimerization of isoprene, leading to a conversion rateabove 50% (Masotti, Henriette; Peiffer, Gilbert; Siv, Chhan; Courbis,Pierre; Sergent, Michelle; Phan Tan Luu, Roger, Bulletin des SocietesChimiques Belges (1991), 100(1), 63-77.)

Thermal dimerization of isoprene at 110-250° in presence ofdinitrocresol as polymerization inhibitor gives high yields of dimersand little polymer (U.S. Pat. No. 4,973,787.) Ni-catalyzed dimerizationof isoprene yields a dimethyl-1,5-cyclooctadiene mixture consisting of80% 1,5-dimethyl-1,5-cyclooctadiene and 20%1,6-dimethyl-1,5-cyclooctadiene (Doppelt, Pascal; Baum, Thomas H.;Ricard, Louis, Inorganic Chemistry (1996), 35(5), 1286-91.) Isoprene isconverted to dimethylcyclooctadienes with a catalytic amt. ofCp*Ru(η4-isoprene)Cl and AgOTf (Itoh, Kenji; Masuda, Katsuyuki;Fukahori, Takahiko; Nakano, Katsumasa; Aoki, Katsuyuki; Nagashima,Hideo, Organometallics (1994), 13(3), 1020-9.) JP59065026A (1984)reported preparation of 1,6-dimethyl-1,5-cyclooctadiene by cyclicdimerization of isoprene in the presence of catalysts comprising Fecarboxylates or β-diketone compounds, organo-Al or Mg compounds, and2,2′-dipyridyl derivatives having electron-donating groups.Dimethylcyclooctadiene was prepared by cyclodimerization of isopreneover 3-component catalysts containing Ni carboxylates or β-ketones,organoaluminum or organomagnesium compounds and substitutedtriphenylphosphite (JP58055434A, 1983.) 1,5-Dimethyl-1,5-cyclooctadienewas prepared by cyclodimerization of isoprene at 100-300° in an inertorganic solvent in the presence of a homogeneous catalyst containingFe(3) salt, organoaluminum compound and an activator (SU615056A1, 1978),in the presence of a homogeneous catalyst containing Ni acetylacetonate,a triarylphosphite and perhydroalumophenolene (SU493-455A1, 1975), inthe presence of a catalyst containing a mixture of a Ni carboxylate orcarboxylate or chelate compounds of Ni and 1-hydroxy-3-carbonylcompounds, trialkylaluminum, dialkylmagnesium or active organo-Mgcompounds obtained from conjugated dienes and Mg, triaryl phosphites andtertiary amines (JP48064049A, 1973), or in the presence of a catalystcomposed of Ni naphthenate, Et₃Al, and tri-o-cresyl phosphate (Suga, K.;Watanabe, S.; Fujita, T.; Shimada, T., Israel Journal of Chemistry(1972), 10(1), 15-18.) U.S. Pat. No. 3,954,665 disclosed dimerization ofisoprene in the presence of reaction products of [(η3-C6H5)NiBr]₂ or[M(NO)₂X]₂ (M=Fe, Co; X═Cl, I, Br) with Fe, Co, or Ni carbonyls.European Patent No. 2411 (1981) disclosed cyclodimerization of isopreneover a Fe(NO)₂Cl-bis(1,5-cyclooctadiene)nickel catalyst at from −5° to+20° to give 1-methyl- and 2-methyl-4-isopropenyl-1-cyclohexene and 1,4-and 2,4-dimethyl-4-vinyl-1-cyclohexene. U.S. Pat. No. 4,189,403disclosed preparation of 1,5-dimethyl-1,5-cyclooctadiene and1,4-dimethyl-4-vinyl-1-cyclohexene by contacting isoprene with a mixedcatalyst of a tris(substituted hydrocarbyl) phosphite, arsenite, orantimonite and a Group VIII metal(0) compound (e.g. Ni acetylacetonate).Jackstell, R.; Grotevendt, A.; Michalik, D.; El Firdoussi, L.; Beller,M. J. Organometallic Chem. (2007) 692(21), 4737-4744 cites the use ofpalladium/carbene catalysts for isoprene dimerization. Bowen, L.;Charernsuk, M.; Wass, D. F. Chem. Commun. (2007) 2835-2837 describes theuse of a chromium N,N-bis(diarylphosphino)amine catalyst for theproduction of linear and cyclic trimers of isoprene.

Isoprene was reportedly dimerized in the presence of a Ni catalyst toyield cis-2-isopropenyl-1-methylvinylcyclobutane (Billups, W. E.; Cross,J. H.; Smith, C. V., Journal of the American Chemical Society (1973),95(10), 3438-9.) The oligomerization of isoprene [78-79-5] catalyzed bynickel naphthenate and isoprenemagnesium in the presence of variousphosphites as electron donors gave cyclic dimers containingdimethylcyclooctadiene [39881-79-3]; in particular1,1,1-tris(hydroxymethyl)propane phosphite [39865-19-5] gavetrimethylcyclododecatriene [39881-80-6] selectively (Suga, Kyoichi;Watanabe, Shoji; Fujita, Tsutomu; Shimada, Takashi, Journal of AppliedChemistry & Biotechnology (1973), 23(2), 131-8.) WO2006/051011 disclosespreparation of trimethylcyclododecatriene, useful in perfumes andfragrances, by the trimerization of isoprene in the presence of acatalyst system comprising Ni and/or Ti, one or more organometalliccompound, and a Group VA compound, and that the reaction is conducted ina hydroxyl group-containing solvent. Ligabue, R. A.; Dupont, J.; deSouza, R. F., Alegre, R. S. J. Mol. Cat. A: Chem. (2001), 169(1-2),11-17, describes the selective dimerization of isoprene to six-membereddimers using an iron nitrosyl catalyst in an ionic liquid. Huchette, D.;Nicole, J.; Petit, F., Tetrahedron Letters (1979), (12), 1035-8,describes the electrochemical generation of an iron nitrosyl catalystand subsequent use for the dimerization of isoprene to cyclohexenedimers. Zakharkin, L. I.; Zhigareva, G. G.; Pryanishnikov, A. P. ZhurnalObshchei Khimii (1987), 57(11), 2551-6, describes thecyclooligomerization of isoprene on complex nickel and iron catalysts.

The highly pure isoprene starting compositions described herein arechemically transformed using each catalyst systems and reactionconditions disclosed in the references cited. Other catalysts andreaction conditions known in the art such as catalysts and reactionconditions applied to chemical transformations of 1,3-butadiene can beadapted to the isoprene starting compositions by one skilled in the art.

Dimerization and Trimerization

In some embodiments, a commercially beneficial amount of highly pureisoprene starting material undergoes catalytic chemical transformationto give dimers and trimers. Catalyst systems are identified usingmethods known in the art. Preferred catalysts include those known toconvert isoprene to dimers and trimers with high efficiency. Examplesinclude those based upon Palladium, Nickel, Cobalt, Iron and Chromium.

In some embodiments, a commercially beneficial amount of highly pureisoprene starting material undergoes thermal or catalytic dimerization.The isoprene starting composition is converted to a mixture of dimersthat includes unsaturated 6 and 8-membered rings by heating the startingcomposition under pressure. In some embodiments, the starting isoprenecomposition is heated to over or about 100, 125, 150, 175, 200, 225 or250° C. under pressure. In some embodiments, the starting isoprenecomposition is heated in the presence of an antioxidant (e.g.2,6-di-tert-butyl-4-methylphenol) to prevent radical-mediatedpolymerization. In some embodiments, the reaction is accelerated by oneor more catalysts selected from catalysts known in the art forcatalyzing cyclic dimerization of isoprene, e.g., iron nitrosyl halidecatalysts described in U.S. Pat. Nos. 4,144,278, 4,181,707, 5,545,789and European patent EP0397266A2. The use of iron nitrosyl catalysts inionic liquids is described by Ligabue et al. (2001) J. Mol. Catalysis.A: Chemical, 169 (2), 11-17. Examples of catalysts include but are notlimited to Fe(NO)₂Cl₂ and Cr³⁺-montmorillonite. In another example, anisoprene starting composition is converted to C₁₀ cyclic dimers (e.g. amixture of dimethyl-cyclooctadienes) using a ruthenium catalyst (Itoh,Kenji; Masuda, Katsuyuki; Fukahori, Takahiko; Nakano, Katsumasa; Aoki,Katsuyuki; Nagashima, Hideo, Organometallics (1994), 13(3), 1020-9.)

In a specific embodiment, a isoprene starting composition in a liquidstate is heated to over 100° C. under pressure and in the presence of anantioxidant to produce a mixture of dimers that includes 6 and8-membered rings, for examples, limonene(1-methyl-4-(prop-1-en-2-yl)cyclohex-1-ene),1-methyl-5-(prop-1-en-2-yl)cyclohex-1-ene,1,4-dimethyl-4-vinylcyclohexene, 2,4-dimethyl-4-vinylcyclohexene,1,5-dimethylcycloocta-1,5-diene, 1,6-dimethylcycloocta-1,5-diene,2,6-dimethyl-1,3-cyclooctadiene, 2,6-dimethyl-1,4-cyclooctadiene,3,7-dimethyl-1,5-cyclooctadiene, 3,7-dimethyl-1,3-cyclooctadiene,3,6-dimethyl-1,3-cyclooctadiene and other 8-membered ring isoprenedimers as described in Organometallics (1994), 13(3), 1020-9. Allstereoisomers of these compounds are contemplated. Conversion ofisoprene to dimers is best performed in the absence of oxygen so as toavoid the production of undesirable reaction products.

In some embodiments, a commercially beneficial amount of highly pureisoprene starting material undergoes photo-dimerization to give4-membered ring dimers. The isoprene starting composition is convertedby light irradiation to a mixture comprising one or more of1,2-di(prop-1-en-2-yl)cyclobutane, 1,3-di(prop-1-en-2-yl)cyclobutane,1-methyl-1-vinyl-3-(prop-1-en-2-yl)cyclobutane,1-methyl-1-vinyl-2-(prop-1-en-2-yl)cyclobutane,1,3-dimethyl-1,3-divinylcyclobutane, 1,2-dimethyl-1,2-divinylcyclobutaneand stereoisomers thereof. In some embodiments, the highly pure isoprenestarting material is dimerized in the presence of a catalyst (e.g. anickel catalyst) or a photosensitizer (e.g. benzophenone) to yield4-membered ring dimers, e.g. cis-2-isopropenyl-1-methylvinylcyclobutane,in addition to 6- and 8-membered rings [See: Hammond, J. S.; Turro, N.J.; Liu, R. S. H. (1963) “Mechanisms of Photochemical Reactions inSolution. XVI. Photosensitized Dimerization of Conjugated Dienes. J.Org. Chem., 28, 3297-3303.]

In some embodiments, a commercially beneficial amount of highly pureisoprene starting material is converted to a cyclic trimer in thepresence of a catalyst system. In some embodiments, the cyclic trimer istrimethylcyclododecatriene. In some embodiments, the catalyst systemcomprises a nickel catalyst. In some embodiments, the catalyst systemcomprises a titanium compound. In some embodiments, the catalyst systemcomprises a nickel catalyst and a titanium compound. In someembodiments, the catalyst system comprises one or more organometalliccompound and a Group VA compound. In some embodiments, the catalytictransformation is conducted in a hydroxyl group-containing solvent. In aparticular embodiment, a starting isoprene composition is converted totrimethylcyclododecatriene in the presence of a catalyst systemcomprising Ni and/or Ti, one or more organometallic compound, and aGroup VA compound, and that the reaction is conducted in a hydroxylgroup-containing solvent.

In some embodiments, a commercially beneficial amount of highly pureisoprene starting material undergoes thermal or catalytic conversion tohydrocarbons in the C7 to C14 range by treatment with a C2 to C5unsaturated hydrocarbon. The C2 to C5 hydrocarbon can be an alkene, adiene or an alkyne. For example, in some embodiments, isoprene iscontacted with ethylene and an appropriate catalyst to produce C7compounds. In another embodiment, isoprene is contacted with1,3-butadiene to form cyclic C9 hydrocarbons. Hydrogenation of these C7to C14 hydrocarbons results in compositions suitable for use as jetfuels and other aviation fuels. In yet another embodiment, unsaturatedC7 to C14 hydrocarbons derived from isoprene and one or more unsaturatedhydrocarbons undergo dehydrogenation to form aromatic derivativessuitable for use as jet fuels and other aviation fuels, or asblendstocks for such fuels.

In some embodiments, a commercially beneficial amount of highly pureisoprene starting material is converted to a mixture of linear dimersand trimers using a catalyst system in alcohol solvents. This reactionalso produces alkoxy derivatives when performed in methanol or ethanol.In some embodiments, the catalyst system is a palladium-based catalystsystem, e.g., a palladium acetylacetonate-triphenylphosphine system or apalladium acetate-triphenylphosphine system. In some embodiments, thealcohol solvent is methanol, ethanol or isopropyl alcohol. The nature ofthe products depends on the catalyst and the solvent used. For example,when a palladium-based catalyst system, e.g., Pd(acac)₂-Ph₃P orPd(OAc)₂-Ph₃P, is used in isopropyl alcohol solvent, linear dimers ofisoprene is formed, e.g. 2,7-dimethyl-1,3,7-octatriene and2,7-dimethyl-2,4,6-octatriene. When this reaction is performed inmethanol, methoxydimethyloctadienes (e.g.,1-methoxy-2,7-dimethyl-2,7-octadiene and3-methoxy-2,7-dimethyl-1,7-octadiene) are formed in addition to linearisoprene dimers such as dimethyloctatrienes. Some reactions producelinear trimers of isoprene, such as a-farnesene(3,7,11-trimethyl-1,3,6,10-dodecatetraene), β-farnesene(7,11-dimethyl-3-methylene-1,6,10-dodecatriene) and other positionalisomers (e.g. from tail to tail and head to head addition of isopreneunits). In another example, isoprene is converted to linear and cyclicC15 trimers using a chromium N,N-bis(diarylphosphino)amine catalyst(Bowen, L.; Charernsuk, M.; Wass, D. F. Chem. Commun. (2007) 2835-2837.)

Conversion to Oxygenates

In some embodiments, a commercially beneficial amount of highly pureisoprene starting material is converted to fuel oxygenates by reactionwith ethanol and other alcohols in the presence of an acid catalyst. Inone embodiment, the acid catalyst is sulfuric acid. In anotherembodiment, the acid catalyst is a solid phase sulfuric acid (e.g.,Dowex Marathon®). Other catalysts include both liquid and solid-phasefluorosulfonic acids, for example, trifluoromethanesulfonic acid andNafion-H (DuPont). Zeolite catalysts can also been used, for examplebeta-zeolite, under conditions similar to those described by Hensel etal. [Hensen, K.; Mahaim, C.; Holderich, W. F., Applied Catalysis A:General (1997) 140(2), 311-329.] for the methoxylation of limonene andrelated monoterpenes. In some embodiments, a highly pure isoprenestarting material is converted to alcohols and esters by a hydroxylationesterification process or other known reaction of alkenes in the art,for example peroxidation to epoxides with peracids such as peraceticacid and 3-chloroperbenzoic acid; and hydration to give alcohols anddiols with i) water and acid catalysts and ii) hydroboration methods.Such reactions are described in, for example, Michael B. Smith and JerryMarch, Advanced Organic Chemistry: Reactions, Mechanisms, and Structure,Sixth Edition, John Wiley & Sons, 2007. FIGS. 3 and 4 show examples ofalcohols and oxygenates that can be produced from the starting isoprenecompositions.

Partial Hydrogenation

In some embodiments, a commercially beneficial amount of highly pureisoprene starting material is partially hydrogenated to a mono-olefin(e.g. 2-methylbut-1-ene, 3-methyl-but-1-ene and 2-methylbut-2-ene). Insome embodiments, the mono-olefin undergoes dimerization or reactionwith other olefins using traditional hydrocarbon cationic catalysis,such as that used to covert isobutylene to isooctane. See, for example,H. M. Lybarger. Isoprene in Kirk-Othmer Encyclopedia of ChemicalTechnology, 4th ed., Wiley, New York (1995), 14, 934-952. In somepreferred embodiments, the highly pure isoprene starting material is abioisoprene composition.

In some embodiments, a commercially beneficial amount of highly pureisoprene starting material undergoes partial hydrogenation to formmono-olefins (e.g. 2-methylbut-1-ene, 3-methyl-but-1-ene and2-methylbut-2-ene). In some preferred embodiments, the highly pureisoprene starting material is a bioisoprene composition. In someembodiments, partial hydrogenation of the isoprene starting materialproduces high yields of mono-olefins with minimal conversion toisopentane and low residual isoprene levels. In some embodiments, amixture of mono-olefin and isopentane is produced, with low levels ofresidual isoprene. In some embodiments, the partial hydrogenation isselective hydrogenation where a particular mono-olefin such as2-methylbut-2-ene, 2-methylbut-1-ene, or 3-methylbut-1-ene ispreferentially produced. Preferred hydrogenation catalysts give highcatalytic activity, maintain catalytic activity over time, and arehighly selective for the conversion of isoprene to mono-olefins.

Suitable catalysts for partial hydrogenation of the isoprene startingcomposition may contain a platinum-group metal such as platinum,palladium, rhodium, or ruthenium, or a transition metal such as nickel,cobalt, copper, iron, molybdenum, or a noble metal such as silver orgold in elemental form, or a salt or complex with organic and inorganicligands. Alloys of these metals can also be used in some circumatances.These metals and their derivatives can be pure or mixed with otheractive and inert materials. Catalysts may be adsorbed to a supportmaterial (such as activated carbon, alumina, or silica) in order tomaximize the effective surface area. The physical morphology ofhydrogenation catalysts is known to have a considerable influence ontheir performance. Preferred hydrogenation catalysts includeheterogeneous palladium catalysts such as palladium on carbon (Pd/C),palladium on alumina (Pd/Al₂O₃), or palladium on silica (Pd/SiO₂) ingrades ranging from 0.1% Pd to 20% Pd (w/w) relative to the supportmaterial. An example of a suitable selective hydrogenation catalyst isLD 2773, a sulfur tolerant promoted Pd on alumina (Axens,Rueil-Malmaison Cedex, France). Partial hydrogenation catalysts thatallow the conversion of isoprene into isoamylenes with minimalconversion to isopentane include the Lindlar catalyst (Pd/BaSO₄ treatedwith quinoline), Pd/C treated with the triphenyl derivative of a group15 element (N, P, As), Pd/C treated with a sulfur-containing compound,molybdenum sulfide, and Pd/Fe alloys. One preferred catalyst comprisespalladium adsorbed to egg-shell alumina (d-Al₂O₃). Catalysts used in therefining industry for the removal of diolefins and alkynes frompyrolysis gasoline are particularly preferred, for example Ni/Al₂O₃ andPd/Al₂O₃ based catalysts. Another suitable class of catalyst for theconversion of isoprene to isoamylenes are those used in industry for thehydrotreating of pyrolysis gasoline. See for example U.S. Pat. Nos.7,014,750 and 6,9949,686, and references cited therein. In general, thecatalysts used in pyrolysis gas hydrotreament allow for selectiveconversion of acetylyenes and diolefins (e.g. isoprene and piperylenes)into monoolefins.

The hydrogen source can be hydrogen gas or a hydrogen source including,but not limited to, hydrogen gas (H₂), formic acid, hydrazine, orisopropanol. The hydrogen source can be either chemically orbiologically derived. In some embodiments the hydrogen used forhydrogenation is co-produced with isoprene during fermentation. Thehydrogenation can be performed at hydrogen pressures ranging from 0.5atm to 200 atm, or higher. The temperature can range from 0° C. to 200°C.

Products from partial hydrogenation of a bioisoprene composition areexpected to contain certain impurities originally present in thestarting isoprene composition and/or hydrogenated derivatives of theimputities such as acetone, ethanol, amyl alcohol, ethyl acetate,isoamyl acetate, methyl ethyl ketone and other saturated polarimpurities.

In some embodiments, the isoprene starting composition undergoes partialhydrogenation or selective hydrogenation in the presence of a palladiumcatalyst. For example, palladium catalysts, such as Pd/CaCO₃, Pd/BaSO₄,Pd/C, Pd black, Pd/SiO₃, Pd/Al₂O₃, or Pd/SiO₂, were shown to convertisoprene to mono-olefins with a selectivity of greater than 95% overfully reduced C5 alkanes. Use of these palladium catalysts gave amixture of mono-olefin products, with 5% Pd/CaCO₃ having the greatestselectivity for 3-methylbut-1-ene and 5% Pd/SiO₂ having the greatestselectivity for 2-methylbut-2-ene. (See G. C. Bond and A. F. Rawle. J.Mol. Catalysis. A: Chemical 109 (1996) 261-271.) Bond and Rawle havealso shown that palladium-gold and palladium-silver catalysts (e.g.,Pd—Au/SiO₂ and Pd—Ag/SiO₂) have high selectivity for reducing isopreneto mono-olefins over fully reduced C5 alkanes. In some embodiments,silica-supported polyamidoamine (PAMAM) dendrimer-palladium complexeshave been used to selectively catalyze the reduction of cyclic andacyclic dienes to a mixture of mono-olefin isomers. Selectivity for thevarious mono-olefin isomers was dependent on the precise PAMAM ligandused in the catalyst. (See P. P. Zweni and H. Alper. Adv. Synth. Catal.348 (2006) 725-731.) In some embodiments, reduction of isoprene tomono-olefins can be carried out using a Group VIB metal on an inorganicsupport (e.g., a metal zeolite) as a catalyst. For example, a Mo/Al₂O₃catalyst was used to selectively reduce 1,3-butadiene to the respectivemono-olefins. (See U.S. Pat. No. 6,235,954 B1.) In some embodiment,isoprene can be reduced to mono-olefins using a Group VIII metalcatalyst promoted by a metal from Group IB, VIIB, VIIB, or zinc toreduce poisoning of the catalyst. (See U.S. Pat. No. 6,949,686 B2). Insome embodiments, isoprene can be reduced to mono-olefins using amonolithic catalyst bed, which may be in a honeycomb configuration.Catalyst support materials for the monolithic catalyst bed may includemetals such as nickel, platinum, palladium, rhodium, ruthenium, silver,iron, copper, cobalt, chromium, iridium, tin, and alloys or mixturesthereof. (See U.S. Pat. No. 7,014,750 B2). In some embodiments, isoprenecan be reduced to mono-olefins using an eggshell Pd/d-Al₂O₃ catalyst,particularly if the reaction is free of water. The eggshell Pd/d-Al₂O₃catalyst is selective for mono-olefins over fully reduced alkanes and2-methylbut-2-ene is the thermodynamically favorable isomer. (See J.-R.Chang and C.-H. Cheng. Ind. Eng. Chem. Res. 36 (1997) 4094-4099.)

Light olefins (C3-C6) can be readily dimerized using acid catalysts togive higher olefins (C6-C12) also known as dimate. For example, theconversion of isobutylene (2-methylpropene) to isooctene is welldescribed in the art using a variety of acid catalysts includingsulfuric, phosphoric and other mineral acids, sulfonic acids,fluorosulfonic acids, zeolites and acidic clays.

In some embodiments, the isoprene starting composition undergoes partialhydrogenation or selective hydrogenation to form a mono-olefin, and themono-olefin undergoes dimerization or reaction with other olefins usingtraditional hydrocarbon cationic catalysis, such as that used to covertisobutylene to isooctane, e.g. sulfuric, phosphoric and other mineralacids, sulfonic acids, fluorosulfonic acids, zeolites and acidic clays.See, for example, H. M. Lybarger. Isoprene in Kirk-Othmer Encyclopediaof Chemical Technology, 4th ed., Wiley, New York (1995), 14, 934-952. Insome preferred embodiments, the highly pure isoprene starting materialis a bioisoprene composition. Acid catalyst in both liquid and solidforms can be used. Acid resins are preferred and include Amberlyst 15,35, XE586, XN1010 (Rohm and Haas) and similar acidic ion-exchangeresins. Acidic molecular sieves are also preferred catalysts including amedium-pore acid molecular sieve such as ZSM-5, ferrierite, ZSM-22 andZSM-23.

In some embodiments, the isoprene starting composition undergoes partialhydrogenation or selective hydrogenation to form isoamylenes. In someembodiments, the isoamylenes are dimerized to give C10 dimates such asisodecenes. In some embodiments, the isoamylenes are dimerized with anolefin such as propylene, butane or isobutene to give C8-C10 dimates. Insome embodiments, the mono-olefin undergoes dimerization using a resincatalyst (e.g., Amberlyst, Amberlyst 35, Amberlyst 15, Amberlyst XN1010,Amberlyst XE586) to produce diisoamylenes. Use of such resin catalystshas been shown to minimize cracking and further oligomerizationreactions, and under optimal conditions the resin catalysts provideselectivity for dimers of greater than 92%, with trimer formation ofless than 8%. See, for example, M. Marchionna et al. Catalysis Today 65(2001) 397-403. In some embodiments, the mono-olefin undergoesdimerization using a catalytic material containing an acidic mesoporousmolecular sieve, such as a mesoporous sieve embedded in a zeolitestructure, ZSM-5, ferrierite, ZSM-22, or ZSM-23. In a particularembodiment, the catalytic material is thermally stable at hightemperatures (e.g., at least 900° C.). In another particular embodiment,the mono-olefin is substantially free of multi-unsaturated hydrocarbons,such as isoprene. Use of a catalytic material containing an acidicmesoporous molecular sieve for dimerization can lead to selectivity fordimers in excess of 80%. See, for example, US 2007/0191662 A1. In someembodiments, the mono-olefin undergoes dimerization using a solid acidiccatalyst (e.g., a solid phosphoric acid catalyst, acidic ion exchangeresins). Selectivity for dimers using a solid phosphoric acid catalystcan be at least 75%, at least 85% or at least 90%. See, for example, US2009/0099400 A1 and U.S. Pat. No. 6,660,898 B1.

Efficient dimerization can sometimes require the presence of a polarcomponent(s) such as water and oxygenated compound in the feed stream.Examples include alcohols such as methanol, ethanol and t-butanol,ethers such as methyl t-butyl ether (MTBE) and methyl t-amyl ether(TAME) or an ester such as C1 to C5 acetates. In some embodiments,isoamylenes can be converted to ethers such as TAME by treatment withalcohols in the presence of an acid catalyst.

In some embodiments, the C10 dimers of mono-olefins produced by any ofthe methods described herein can be reduced to fully saturated C10alkylates (e.g., by hydrogenation). In a particular embodiment, isopreneor a mono-olefin can be reduced to an alkylate using a catalystcontaining an acid component, such as sulfuric acid, a fluorosulfonicacid, a perhaloalkylsulfonic acid, an ionic liquid, or a mixture ofBronsted acids and Lewis acids, mixed with a polymer component, such asa polyacrylate. (See US 2010/0094072 A1).

In some embodiments, a commercially beneficial amount of highly pureisoprene starting material undergoes oligomerization in the presence ofan acid catalysts to produce dimers, trimers, higher oligomers, aromaticproducts, and/or polymeric products. Kinetic control of the reaction canfavor certain products, for example lower oligomers, although gumformation and coking are known issues leading to catalyst deactivation.

Exemplary Unsaturated Isoprene Derivatives

In some embodiments, the compositions and systems for producing a fuelconstituent from isoprene further comprise an unsaturated hydrocarbon oroxygenate intermediate for chemical transformation of an isoprenestarting composition to a fuel constituent. In some embodiment, theunsaturated hydrocarbon intermediate comprise one or more unsaturateddimers of isoprene selected from the group consisting of1,2-di(prop-1-en-2-yl)cyclobutane, 1,3-di(prop-1-en-2-yl)cyclobutane,1-methyl-1-vinyl-3-(prop-1-en-2-yl)cyclobutane,1-methyl-1-vinyl-2-(prop-1-en-2-yl)cyclobutane,1,3-dimethyl-1,3-divinylcyclobutane,1,2-dimethyl-1,2-divinylcyclobutane,1-methyl-4-(prop-1-en-2-yl)cyclohex-1-ene),1-methyl-5-(prop-1-en-2-yl)cyclohex-1-ene,1,4-dimethyl-4-vinylcyclohexene, 2,4-dimethyl-4-vinylcyclohexene,1,5-dimethylcycloocta-1,5-diene, 1,6-dimethylcycloocta-1,5-diene,1,4-dimethyl-4-vinyl-1-cyclohexene, 2,4-dimethyl-4-vinyl-1-cyclohexene,2,7-dimethyl-1,3,7-octatriene, 2,7-dimethyl-2,4,6-octatriene,2,6-dimethyl-1,3-cyclooctadiene, 2,6-dimethyl-1,4-cyclooctadiene,3,7-dimethyl-1,5-cyclooctadiene, 3,7-dimethyl-1,3-cyclooctadiene and3,6-dimethyl-1,3-cyclooctadiene. In some embodiments, the unsaturatedhydrocarbon intermediate comprises one or more unsaturated trimers suchas a-farnesene, β-farnesene, trimethylcyclododecatrienes (e.g.1,5,9-trimethyl-(1E,5E,9E)-cyclododecatriene and positional andgeometric isomers thereof) and trimethyldodecatetraenes and the like. Insome embodiments, the unsaturated oxygenate intermediate comprise one ormore unsaturated methyl ethers such as1-methoxy-2,7-dimethyl-2,7-octadiene and3-methoxy-2,7-dimethyl-1,7-octadiene and the like. In some embodiments,the unsaturated oxygenate intermediate comprise one or more unsaturatedethyl ethers such as ethyl 3-methyl-3-butenyl ether, ethyl1,1-dimethyl-2-propenyl ether, ethyl 1,2-dimethyl-2-propenyl ether,ethyl 2-methyl-3-butenyl ether and the like. In some embodiments, theunsaturated oxygenate intermediate comprise one or more unsaturatedalcohols such as 3-methyl-3-buten-1-ol, 2-methyl-3-buten-2-ol,3-methyl-3-buten-2-ol, 2-methyl-3-buten-1-ol and the like. In someembodiments, the unsaturated oxygenate intermediate comprise one or moreunsaturated esters such as 3-methyl-3-buten-1-yl acetate,2-methyl-3-buten-2-yl acetate, 3-methyl-3-buten-2-yl acetate,2-methyl-3-buten-1-yl acetate and esters of other C₃-C₁₈ aliphaticcarboxylic acids. In some embodiment, the unsaturated hydrocarbonintermediate comprise one or more isoamylenes, e.g. 2-methylbut-1-ene,3-methyl-but-1-ene and 2-methylbut-2-ene. In some embodiments, theunsaturated hydrocarbon intermediate comprise on or more diisoamylenesderived from dimerization of isoamylenes.

Exemplary Hydrogenation of Unsaturated Intermediates

The unsaturated isoprene derivatives are subject hydrogenation in thepresence of a hydrogenation catalyst to produce saturated compounds. Thesaturated compounds are characterized and their values as fuels areassessed. In some embodiments, the hydrogen source for hydrogenation ishydrogen gas. In some embodiments, the hydrogen gas is co-produced withthe bioisoprene as described in U.S. provisional patent application No.61/141,652, filed on Dec. 30, 2008 and US 2009/0203102 A1. In someembodiments, the hydrogenation catalyst is a palladium based catalystsuch as Pd/C (e.g. 5% (wt.) Pd/C). In some embodiments, thehydrogenation catalyst is a Raney Nickel catalyst. In some embodiments,the hydrogenation catalyst is a homogenous catalyst such as ruthenium orrhodium based homogenous hydrogenation catalysts. In some embodiments,unsaturated isoprene dimers and trimers are hydrogenated to producesaturated C10 and C15 hydrocarbons suitable for making fuels. In someembodiments, unsaturated cyclic dimers are hydrogenated to producesaturated cyclic C10 hydrocarbons such as 1,2-bis(isopropyl)cyclobutane,1,2-bis(isopropyl)cyclobutane, 1-methyl-4-isopropylcyclohexane,1-methyl-3-isopropylcyclohexane, 1-ethyl-1,4-dimethylcyclohexane,1-ethyl-1,3-dimethylcyclohexane, 1,5-dimethylcyclooctane and1,4-dimethylcyclooctane. In some embodiments, unsaturated cyclic trimersare hydrogenated to produce saturated cyclic C15 hydrocarbons such as1,5,9-trimethylcyclododecane and 1,5,10-trimethylcyclododecane (seeScheme VII). In some embodiments, unsaturated linear dimers arehydrogenated to produce saturated aliphatic C10 hydrocarbons such as2,6-dimethyloctane, 2,7-dimethyloctane and 3,6-dimethyloctane. In someembodiments, unsaturated linear trimers are hydrogenated to producesaturated aliphatic C15 hydrocarbons such as 2,6,10-trimethyldodecane,2,7,10-trimethyldodecane and 3,7,10-trimethyldodecane (see Scheme VIII).In some embodiments, the product of the dimerization of isoamylenes(diamylenes or C10 dimates) are fully hydrogenated to isoparaffins (e.g.2,3,4,4-tetramethylhexane, 2,2,3,4-tetramethylhexane,2,3,3,4-tetramethylhexane and 3,3,5-trimethylheptane) (see SchemeVIIIa). In some embodiments, a commercially beneficial amount of highlypure isoprene starting composition is hydrogenated to produce a productcomprising 2-methylbutane. In some embodiments, the unsaturated isoprenehydroxylates are hydrogenated to produce saturated hydroxylates such asC5 alcohols and diols (e.g. 3-methyl-butan-1-ol, 2-methyl-butan-1-ol and2-methyl-butan-2-ol, 3-methyl-butan-1,3-diol and2-methyl-butan-2,3-diol), C-10 alcohols and diols (e.g.3,7-dimethyloctan-1-ol, 2,7-dimethyloctan-1-ol, 2,7-dimethyloctan-2-oland 2,7-dimethyloctan-2,7-diol) and cyclic C-10 alcohols (e.g.2-(4-methylcyclohexyl)propan-2-ol, 2-(4-methylcyclohexyl)propan-1-ol,2-(1,4-dimethylcyclohexyl)ethanol and 4-ethyl-1,4-dimethylcyclohexanol)(see Scheme IX). In some embodiments, the unsaturated isopreneoxygenates are hydrogenated to produce saturated ethers such as1,3-diethoxy-3-methylbutane, 1-ethoxy-3-methylbutane,1-methoxy-2,7-dimethyloctane and 3-methoxy-2,7-dimethyloctane (seeScheme X). It is understood that when an alkene moiety is hydrogenated,one or more stereo isomers are produced. The relative ratios between thestereo isomers depend on the reaction conditions and the catalysts used.When applicable, each and every stereo isomer is intended for thesaturated hydrocarbons and oxygenates described herein.

In some embodiment, the fuel constituent produced by chemicaltransformation of a commercially beneficial amount of highly pureisoprene starting composition comprises saturated isoprene derivatives.In some embodiments, the fuel constituent comprises saturated C10 andC15 hydrocarbons derived from isoprene. In some embodiments, the fuelconstituent comprises one or more saturated cyclic C10 hydrocarbonsselected from the group consisting of 1,2-bis(isopropyl)cyclobutane,1,2-bis(isopropyl)cyclobutane, 1-methyl-4-isopropylcyclohexane,1-methyl-3-isopropylcyclohexane, 1-ethyl-1,4-dimethylcyclohexane,1-ethyl-1,3-dimethylcyclohexane, 1,5-dimethylcyclooctane and1,4-dimethylcyclooctane. In some embodiments, the fuel constituentcomprises one or more saturated cyclic C15 hydrocarbons selected fromthe group consisting of 1,5,9-trimethylcyclododecane and1,5,10-trimethylcyclododecane. In some embodiments, the fuel constituentcomprises one or more saturated aliphatic C10 hydrocarbons selected fromthe group consisting of 2,6-dimethyloctane, 2,7-dimethyloctane and3,6-dimethyloctane. In some embodiments, the fuel constituent comprisesone or more saturated aliphatic C15 hydrocarbons selected from the groupconsisting of 2,6,10-trimethyldodecane, 2,7,10-trimethyldodecane and3,7,10-trimethyldodecane. In some embodiments, the fuel constituentcomprises 2-methylbutane. In some embodiments, the fuel constituentcomprises one or more isoparaffins selected from the group consisting of2,3,4,4-tetramethylhexane, 2,2,3,4-tetramethylhexane,2,3,3,4-tetramethylhexane and 3,3,5-trimethylheptane. In someembodiments, the fuel constituent comprises one or more saturatedhydroxylates selected from the group consisting of 3-methyl-butan-1-ol,2-methyl-butan-1-ol and 2-methyl-butan-2-ol, 3-methyl-butan-1,3-diol,2-methyl-butan-2,3-diol, 3,7-dimethyloctan-1-ol, 2,7-dimethyloctan-1-ol,2,7-dimethyloctan-2-ol, 2,7-dimethyloctan-2,7-diol,2-(4-methylcyclohexyl)propan-2-ol, 2-(4-methylcyclohexyl)propan-1-ol,2-(1,4-dimethylcyclohexyl)ethanol and 4-ethyl-1,4-dimethylcyclohexanol.In some embodiments, the fuel constituent comprises one or moresaturated ethers selected from the group consisting of1,3-diethoxy-3-methylbutane, 1-ethoxy-3-methylbutane,1-methoxy-2,7-dimethyloctane and 3-methoxy-2,7-dimethyloctane. In someembodiments, the fuel constituent comprises one or more oxygenates ofisoprene selected from the group consisting of 3-methyltetrahydrofuran,3-methyl-2-butanone and isopentyl acetate.

In some embodiments, the compositions and systems for producing a fuelconstituent from isoprene further comprise catalysts for catalyzing thechemical transformation of an isoprene starting composition to a fuelconstituent or an intermediate for making a fuel constituent. In someembodiments, the compositions and systems for producing a fuelconstituent from isoprene further comprise catalysts for catalyzinghydrogenation of an unsaturated intermediate to produce a saturated fuelconstituent. In some embodiments, the catalyst is any catalyst describedor a combination of one or more of the catalyst described herein.

Method and/or Processes for Producing Fuels

The invention provides methods and/or processes for producing a fuelconstituent from isoprene comprising: (a) obtaining a commerciallybeneficial amount of highly pure isoprene; and (b) chemicallytransforming at least a portion of the commercially beneficial amount ofhighly pure isoprene to a fuel constituent. In one embodiment, a highlypure isoprene composition is transformed to a fuel component in acontinuous chemical process. In another embodiment, a highly pureisoprene is further purified before chemical transformation to a fuelcomposition. In yet another embodiment, a highly pure isoprene ischemically transformed to an intermediate composition; the intermediatecomposition undergoes further chemical transformations to produce a fuelor a fuel component. In a further embodiment, the fuel componentproduced is mixed with a petroleum distillate and other optionaladditives to make a fuel. In some preferred embodiments, the highly pureisoprene is a bioisoprene composition.

In some embodiments, the method for producing a fuel constituent fromisoprene comprises obtaining a commercially beneficial amount of highlypure isoprene composition. In some embodiments, the highly pure isoprenecomposition useful in the invention comprises greater than or about 2,5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600,700, 800, 900, or 1000 mg of isoprene. In some embodiments, the highlypure isoprene composition comprises greater than or about 2, 5, 10, 20,30, 40, 50, 60, 70, 80, 90, 100 g of isoprene. In some embodiments, thestarting isoprene composition comprises greater than or about 0.2, 0.5,1, 2, 5, 10, 20, 50, 100, 200, 500, 1000 kg of isoprene.

In some embodiments, the method for producing a fuel constituent fromisoprene comprises obtaining a commercially beneficial amount of highlypure isoprene composition. In some embodiments, the highly pure isoprenestarting composition comprises greater than or about 98.0, 98.5, 99.0,99.5, or 100% isoprene by weight compared to the total weight of all C5hydrocarbons in the starting composition. In some embodiments, thehighly pure isoprene composition useful in the invention comprisesgreater than or about 99.90, 99.92, 99.94, 99.96, 99.98, or 100%isoprene by weight compared to the total weight of all C5 hydrocarbonsin the composition. In some embodiments, the highly pure isoprenecomposition comprises less than or about 2.0, 1.5, 1.0, 0.5, 0.2, 0.12,0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001,0.00005, or 0.00001% C5 hydrocarbons other than isoprene (such1,3-cyclopentadiene, cis-1,3-pentadiene, trans-1,3-pentadiene,1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene, 2-methyl-1-butene,3-methyl-1-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, orcis-pent-3-ene-1-yne) by weight compared to the total weight of all C5hydrocarbons in the composition. In some embodiments, the highly pureisoprene composition has less than or about 2.0, 1.5, 1.0, 0.5, 0.2,0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001,0.00005, or 0.00001% for 1,3-cyclopentadiene, cis-1,3-pentadiene,trans-1,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene,2-methyl-1-butene, 3-methyl-1-butyne, pent-4-ene-1-yne,trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne by weight compared tothe total weight of all C5 hydrocarbons in the composition. Inparticular embodiments, the highly pure isoprene composition has greaterthan about 2 mg of isoprene and has greater than or about 98.0, 98.5,99.0, 99.5, 99.90, 99.92, 99.94, 99.96, 99.98, or 100% isoprene byweight compared to the total weight of all C5 hydrocarbons in thecomposition.

In some embodiments, the method for producing a fuel constituent fromisoprene comprises obtaining a commercially beneficial amount of highlypure isoprene composition. In some embodiments, the highly pure isoprenecomposition useful in the invention has less than or about 50, 40, 30,20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 μg/L of a compound thatinhibits the polymerization of isoprene for any compound in thecomposition that inhibits the polymerization of isoprene. In particularembodiments, the composition also has greater than about 2 mg ofisoprene.

In some embodiments, the method for producing a fuel constituent fromisoprene comprises obtaining a commercially beneficial amount of highlypure isoprene composition. In some embodiments, the highly pure isoprenecomposition useful in the invention has one or more compounds selectedfrom the group consisting of ethanol, acetone, C5 prenyl alcohols, andisoprenoid compounds with 10 or more carbon atoms. In some embodiments,the highly pure isoprene composition has greater than or about 0.005,0.01, 0.05, 0.1, 0.5, 1, 5, 10, 20, 30, 40, 60, 80, 100, or 120 μg/L ofethanol, acetone, a C5 prenyl alcohol (such as 3-methyl-3-buten-1-ol or3-methyl-2-buten-1-ol), or any two or more of the foregoing. Inparticular embodiments, the highly pure isoprene composition has greaterthan about 2 mg of isoprene and has one or more compounds selected fromthe group consisting of ethanol, acetone, C5 prenyl alcohols, andisoprenoid compounds with 10 or more carbon atoms.

In some embodiments, the method for producing a fuel constituent fromisoprene comprises obtaining a commercially beneficial amount of highlypure isoprene composition. In some embodiments, the highly pure isoprenecomposition useful in the invention includes isoprene and one or moresecond compounds selected from the group consisting of 2-heptanone,6-methyl-5-hepten-2-one, 2,4,5-trimethylpyridine,2,3,5-trimethylpyrazine, citronellal, acetaldehyde, methanethiol, methylacetate, 1-propanol, diacetyl, 2-butanone, 2-methyl-3-buten-2-ol, ethylacetate, 2-methyl-1-propanol, 3-methyl-1-butanal, 3-methyl-2-butanone,1-butanol, 2-pentanone, 3-methyl-1-butanol, ethyl isobutyrate,3-methyl-2-butenal, butyl acetate, 3-methylbutyl acetate,3-methyl-3-buten-1-yl acetate, 3-methyl-2-buten-1-yl acetate,3-hexen-1-ol, 3-hexen-1-yl acetate, limonene, geraniol(trans-3,7-dimethyl-2,6-octadien-1-ol), citronellol(3,7-dimethyl-6-octen-1-ol), (E)-3,7-dimethyl-1,3,6-octatriene,(Z)-3,7-dimethyl-1,3,6-octatriene, and 2,3-cycloheptenolpyridine. Invarious embodiments, the amount of one of these second componentsrelative to the amount of isoprene in units of percentage by weight(i.e., weight of the component divided by the weight of isoprene times100) is at greater than or about 0.01, 0.02, 0.05, 0.1, 0.5, 1, 5, 10,20, 30, 40, 50, 60, 70, 80, 90, 100, or 110% (w/w). In some embodiments,the amount of methanethiol relative to the amount of isoprene in unitsof percentage by weight is at less than 0.01% (w/w).

In some embodiments, the method for producing a fuel constituent fromisoprene comprises obtaining a commercially beneficial amount of highlypure isoprene composition. In some embodiments, the method comprisesobtaining a commercially beneficial amount of highly pure isoprenecomposition from a biological process comprising: (a) culturing cellscomprising a heterologous nucleic acid encoding an isoprene synthasepolypeptide under suitable culture conditions for the production ofisoprene, wherein the cells (i) produce greater than about 400nmole/g_(wcm)/hr of isoprene, (ii) convert more than about 0.002 molarpercent of the carbon that the cells consume from a cell culture mediuminto isoprene, or (iii) have an average volumetric productivity ofisoprene greater than about 0.1 mg/L_(broth)/hr of isoprene, and (b)producing isoprene. In some embodiments, the cells have a heterologousnucleic acid that (i) encodes an isoprene synthase polypeptide, e.g. anaturally-occurring polypeptide from a plant such as Pueraria, and (ii)is operably linked to a promoter, e.g. a T7 promoter. In someembodiments, the cells are cultured in a culture medium that includes acarbon source, such as, but not limited to, a carbohydrate, glycerol,glycerine, dihydroxyacetone, one-carbon source, oil, animal fat, animaloil, fatty acid, lipid, phospholipid, glycerolipid, monoglyceride,diglyceride, triglyceride, renewable carbon source, polypeptide (e.g., amicrobial or plant protein or peptide), yeast extract, component from ayeast extract, or any combination of two or more of the foregoing. Insome embodiments, the cells are cultured under limited glucoseconditions. In some embodiment, the cells further comprise aheterologous nucleic acid encoding an IDI polypeptide. In someembodiments, the cells further comprise a heterologous nucleic acidencoding an MDV pathway polypeptide. In some embodiment, the methodcomprises producing isoprene using the methods described in U.S.provisional patent application Nos. 61/134,094, filed on Jul. 2, 2008,WO 2010/003007, and U.S. patent application Ser. No. 12/335,071, filedDec. 15, 2008 (US 2009/0203102 A1), which are incorporated by referencein their entireties.

In some embodiment, the method comprises obtaining a gas phase (off-gas)produced by cells in culture that produces isoprene. In someembodiments, the cells in culturing produce greater than about 400nmole/g_(wcm)/hr of isoprene. In some embodiments, the gas phasecomprises greater than or about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90,100 μg/L of isoprene when sparged at a rate of 1 vvm. In someembodiments, the volatile organic fraction of the gas phase comprisesgreater than or about 99.90, 99.92, 99.94, 99.96, 99.98, or 100%isoprene by weight compared to the total weight of all C5 hydrocarbonsin the volatile organic fraction. In some embodiments, the volatileorganic fraction of the gas phase comprises less than or about 2.0, 1.5,1.0, 0.5, 0.2, 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001,0.0005, 0.0001, 0.00005, or 0.00001% C5 hydrocarbons other than isoprene(such 1,3-cyclopentadiene, cis-1,3-pentadiene, trans-1,3-pentadiene,1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene, 2-methyl-1-butene,3-methyl-1-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, orcis-pent-3-ene-1-yne) by weight compared to the total weight of all C5hydrocarbons in the volatile organic fraction. In some embodiments, thevolatile organic fraction of the gas phase has less than or about 2.0,1.5, 1.0, 0.5, 0.2, 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005,0.001, 0.0005, 0.0001, 0.00005, or 0.00001% for 1,3-cyclopentadiene,cis-1,3-pentadiene, trans-1,3-pentadiene, 1,4-pentadiene, 1-pentyne,2-pentyne, 1-pentene, 2-methyl-1-butene, 3-methyl-1-butyne,pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne byweight compared to the total weight of all C5 hydrocarbons in thevolatile organic fraction. In particular embodiments, the volatileorganic fraction of the gas phase has greater than about 2 mg ofisoprene and has greater than or about 99.90, 99.92, 99.94, 99.96,99.98, or 100% isoprene by weight compared to the total weight of all C5hydrocarbons in the volatile organic fraction.

In some embodiment, the method comprises obtaining a gas phase (off-gas)produced by cells in culture that produces isoprene. In someembodiments, the volatile organic fraction of the gas phase has lessthan or about 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005μg/L of a compound that inhibits the polymerization of isoprene for anycompound in the volatile organic fraction of the gas phase that inhibitsthe polymerization of isoprene. In particular embodiments, the volatileorganic fraction of the gas phase also has greater than about 2 mg ofisoprene. In some embodiments, the volatile organic fraction of the gasphase has one or more compounds selected from the group consisting ofethanol, acetone, C5 prenyl alcohols, and isoprenoid compounds with 10or more carbon atoms. In some embodiments, the volatile organic fractionof the gas phase has greater than or about 0.005, 0.01, 0.05, 0.1, 0.5,1, 5, 10, 20, 30, 40, 60, 80, 100, or 120 μg/L of ethanol, acetone, a C5prenyl alcohol (such as 3-methyl-3-buten-1-ol or 3-methyl-2-buten-1-ol),or any two or more of the foregoing. In particular embodiments, thevolatile organic fraction of the gas phase has greater than about 2 mgof isoprene and has one or more compounds selected from the groupconsisting of ethanol, acetone, C5 prenyl alcohols, and isoprenoidcompounds with 10 or more carbon atoms. In some embodiments, thevolatile organic fraction of the gas phase has includes isoprene and oneor more second compounds selected from the group consisting of2-heptanone, 6-methyl-5-hepten-2-one, 2,4,5-trimethylpyridine,2,3,5-trimethylpyrazine, citronellal, acetaldehyde, methanethiol, methylacetate, 1-propanol, diacetyl, 2-butanone, 2-methyl-3-buten-2-ol, ethylacetate, 2-methyl-1-propanol, 3-methyl-1-butanal, 3-methyl-2-butanone,1-butanol, 2-pentanone, 3-methyl-1-butanol, ethyl isobutyrate,3-methyl-2-butenal, butyl acetate, 3-methylbutyl acetate,3-methyl-3-buten-1-yl acetate, 3-methyl-2-buten-1-yl acetate,3-hexen-1-ol, 3-hexen-1-yl acetate, limonene, geraniol(trans-3,7-dimethyl-2,6-octadien-1-ol), citronellol(3,7-dimethyl-6-octen-1-ol), (E)-3,7-dimethyl-1,3,6-octatriene,(Z)-3,7-dimethyl-1,3,6-octatriene, and 2,3-cycloheptenolpyridine. Invarious embodiments, the amount of one of these second componentsrelative to amount of isoprene in units of percentage by weight (i.e.,weight of the component divided by the weight of isoprene times 100) isat greater than or about 0.01, 0.02, 0.05, 0.1, 0.5, 1, 5, 10, 20, 30,40, 50, 60, 70, 80, 90, 100, or 110% (w/w) in the volatile organicfraction of the gas phase. In some embodiments, the method furtherincludes recovering the isoprene from the gas phase. For example, theisoprene in the gas phase comprising isoprene can be recovered usingstandard techniques, such as gas stripping, membrane enhancedseparation, fractionation, adsorption/desorption, pervaporation, thermalor vacuum desorption of isoprene from a solid phase, or extraction ofisoprene immobilized or absorbed to a solid phase with a solvent (see,for example, U.S. Pat. Nos. 4,703,007 and 4,570,029, which are eachhereby incorporated by reference in their entireties, particularly withrespect to isoprene recovery and purification methods). In someparticular embodiments, extractive distillation with an alcohol (such asethanol, methanol, propanol, or a combination thereof) is used torecover the isoprene. In some embodiments, the recovery of isopreneinvolves the isolation of isoprene in a liquid form (such as a neatsolution of isoprene or a solution of isoprene in a solvent). Gasstripping involves the removal of isoprene vapor from the fermentationoff-gas stream in a continuous manner. Such removal can be achieved inseveral different ways including, but not limited to, adsorption to asolid phase, partition into a liquid phase, or direct condensation (suchas condensation due to exposure to a condensation coil or do to anincrease in pressure). In some embodiments, membrane enrichment of adilute isoprene vapor stream above the dew point of the vapor resultingin the condensation of liquid isoprene. In some embodiments, therecovered isoprene is compressed and condensed.

The recovery of isoprene may involve one step or multiple steps. In someembodiments, the removal of isoprene vapor from a fermentation off-gasand the conversion of isoprene to a liquid phase are performedsimultaneously. For example, isoprene can be directly condensed from theoff-gas stream to form a liquid. In some embodiments, the removal ofisoprene vapor from the fermentation off-gas and the conversion ofisoprene to a liquid phase are performed sequentially. For example,isoprene may be adsorbed to a solid phase and then extracted from thesolid phase with a solvent.

In some embodiments, a method is provided for producing a cyclic dimerof isoprene comprising heating neat bioisoprene. The cyclic dimers ofisoprene produced may be 6-membered ring dimers (e.g. a [2+4]electrocyclization product such as limonene) or 8-membered ring dimersor a mixture thereof. Examples of 6-membered ring dimers include but notlimited to 1-methyl-4-(prop-1-en-2-yl)cyclohex-1-ene,1-methyl-5-(prop-1-en-2-yl)cyclohex-1-ene,1,4-dimethyl-4-vinylcyclohexene, 2,4-dimethyl-4-vinylcyclohexene and thelike. Examples of 8-membered ring dimers include but not limited to1,5-dimethylcycloocta-1,5-diene, 1,6-dimethylcycloocta-1,5-diene and thelike. In one embodiment, neat biologically produced isoprene isdimerized by heating to a temperature of about 100° C. to about 300° C.,preferably about 150° C. to about 250° C. The pressure is maintained atabout 2 to 3 atm. In another embodiment, a catalyst suitable forcatalyzing dimerization of conjugated dienes may be used. Theproportions of various dimers in the product mixture may be controlledby the catalyst and other reactions conditions. For example, a nickelcatalyst can promote formation of 8-membered ring dimers.

In some embodiments, a method is provided for producing a cyclic dimerof isoprene comprising photo-dimerization of bioisoprene. The cyclicdimer of isoprene produced may be one or more 4-membered ring dimer or amixture thereof. Examples of Examples of 4-membered ring dimers includebut not limited to 1,2-di(prop-1-en-2-yl)cyclobutane,1,3-di(prop-1-en-2-yl)cyclobutane,1-methyl-1-vinyl-3-(prop-1-en-2-yl)cyclobutane,1-methyl-1-vinyl-2-(prop-1-en-2-yl)cyclobutane,1,3-dimethyl-1,3-divinylcyclobutane, 1,2-dimethyl-1,2-divinylcyclobutaneand the like. In one embodiment, neat biologically produced isoprene isdimerized by irradiating with UV light, preferably in presence of aphotosensitizer such as benzophenone.

Isoprene dimers can be hydrogenated to form saturated C₁₀ hydrocarbonsthat can serve as fuels or blended into fuels. In one embodiment, theunsaturated isoprene dimers are subjected to catalytic hydrogenation toproduce partially hydrogenated and/or fully hydrogenated products.Examples of fully hydrogenated products include but not limited to1-methyl-4-isopropylcyclohexane and 1-methyl-5-isopropylcyclohexane,1,4-dimethyl-4-ethylcyclohexane, 2,4-dimethyl-4-ethylcyclohexane,1,2-diisopropylcyclobutane and 1,3-diisopropylcyclobutane,1-methyl-1-vinyl-3-(prop-1-en-2-yl)cyclobutane,1-methyl-1-ethyl-2-(prop-2-yl)cyclobutane,1,3-dimethyl-1,3-ethylcyclobutane, 1,2-dimethyl-1,2-diethylcyclobutane,1,5-dimethylcyclooctane, 1,6-dimethylcyclooctane and the like.

In some embodiments, the method for producing a fuel constituent fromisoprene comprises: (a) obtaining a commercially beneficial amount ofany of the highly pure isoprene starting composition described herein;(b) chemically transforming at least a portion of the highly pureisoprene starting composition to a fuel constituent comprising: (i)heating the highly pure isoprene composition to over or about 100° C.under pressure; (ii) converting at least a portion of the startingisoprene composition to unsaturated cyclic isoprene dimers; and (iii)hydrogenating the unsaturated cyclic isoprene dimers to producesaturated cyclic isoprene dimers. In some embodiments, the startingisoprene composition is heated to over or about 100, 125, 150, 175, 200,225 or 250° C. under pressure. In some embodiments, the startingisoprene composition is heated in the presence of an antioxidant (e.g.2,6-di-tert-butyl-4-methylphenol) to prevent radical-mediatedpolymerization. In one embodiment, the thermal dimerization of isopreneis performed in presence of dinitrocresol as polymerization inhibitor.Suitable antioxidants may be used as polymerization inhibitors. In someembodiments, at least a portion of the starting isoprene composition toa mixture of unsaturated dimers that includes 6 and 8-membered rings,for examples, limonene (1-methyl-4-(prop-1-en-2-yl)cyclohex-1-ene),1-methyl-5-(prop-1-en-2-yl)cyclohex-1-ene,1,4-dimethyl-4-vinylcyclohexene, 2,4-dimethyl-4-vinylcyclohexene,1,5-dimethylcycloocta-1,5-diene, 1,6-dimethylcycloocta-1,5-diene,2,6-dimethyl-1,3-cyclooctadiene, 2,6-dimethyl-1,4-cyclooctadiene,3,7-dimethyl-1,5-cyclooctadiene, 3,7-dimethyl-1,3-cyclooctadiene and3,6-dimethyl-1,3-cyclooctadiene. In some embodiments, greater than orabout 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% ofisoprene in starting isoprene composition is converted to unsaturatedcyclic isoprene dimers. In some embodiments, hydrogenation of theunsaturated cyclic isoprene dimers is catalyzed by a hydrogenationcatalyst such as a palladium based catalyst (e.g. Pd/C, e.g. 5% (wt.)Pd/C.) In some embodiments, the saturated cyclic isoprene dimerscomprise one or more C10 hydrocarbons selected from the group consistingof 1-methyl-4-isopropylcyclohexane, 1-methyl-3-isopropylcyclohexane,1,5-dimethylcyclooctane and 1,4-dimethylcyclooctane. In some preferredembodiments, the starting isoprene composition is a bioisoprenecomposition.

In some embodiments, the method for producing a fuel constituent fromisoprene comprises: (a) obtaining a commercially beneficial amount ofany of the highly pure isoprene starting composition described herein;(b) chemically transforming at least a portion of the highly pureisoprene starting composition to a fuel constituent comprising: (i)contacting the highly pure isoprene composition with a catalyst system;(ii) converting at least a portion of the starting isoprene compositionto unsaturated isoprene dimers and/or trimers; and (iii) hydrogenatingthe unsaturated dimers and/or trimers to produce saturated C10 and/orC15 hydrocarbons. In some embodiments, greater than or about 20, 30, 40,50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% of isoprene in startingisoprene composition is converted to unsaturated dimers and/or trimersof isoprene. In some embodiments, the starting isoprene composition iscontacted with appropriate catalyst systems known in the art to yieldone or more of unsaturated cyclic isoprene dimers selected from thegroup consisting of 1,2-di(prop-1-en-2-yl)cyclobutane,1,3-di(prop-1-en-2-yl)cyclobutane,1-methyl-1-vinyl-3-(prop-1-en-2-yl)cyclobutane,1-methyl-1-vinyl-2-(prop-1-en-2-yl)cyclobutane,1,3-dimethyl-1,3-divinylcyclobutane,1,2-dimethyl-1,2-divinylcyclobutane,1-methyl-4-(prop-1-en-2-yl)cyclohex-1-ene),1-methyl-5-(prop-1-en-2-yl)cyclohex-1-ene,1,4-dimethyl-4-vinylcyclohexene, 2,4-dimethyl-4-vinylcyclohexene,1,5-dimethylcycloocta-1,5-diene, 1,6-dimethylcycloocta-1,5-diene,1,4-dimethyl-4-vinyl-1-cyclohexene, 2,4-dimethyl-4-vinyl-1-cyclohexene,2,7-dimethyl-1,3,7-octatriene, 2,7-dimethyl-2,4,6-octatriene,2,6-dimethyl-1,3-cyclooctadiene, 2,6-dimethyl-1,4-cyclooctadiene,3,7-dimethyl-1,5-cyclooctadiene, 3,7-dimethyl-1,3-cyclooctadiene and3,6-dimethyl-1,3-cyclooctadiene. In some embodiments, the startingisoprene composition is contacted with appropriate catalyst systemsknown in the art to yield one or more of unsaturated cyclic isoprenetrimers such as trimethylcyclododecatrienes and trimethyldodecatetraenesand the like. In some embodiments, the starting isoprene composition iscontacted with appropriate catalyst systems known in the art to yieldone or more of unsaturated linear dimers and/or trimers of isoprene suchas dimethyloctatrienes (e.g. 2,7-dimethyl-1,3,7-octatriene and2,7-dimethyl-2,4,6-octatriene) and trimethyldodecatetraenes (e.g.2,6,10-trimethyl-1,5,9,11-dodecatetraene, a-farnesene and β-farnesene)and the like. In some preferred embodiments, the starting isoprenecomposition is a bioisoprene composition.

In some embodiments, the method for producing a fuel constituent fromisoprene comprises contacting the highly pure isoprene composition witha catalyst system to convert at least a portion of the starting isoprenecomposition to unsaturated isoprene dimers and/or trimers. In someembodiments, the catalyst system comprise a Ni, Fe or Co catalyst andthe isoprene in the starting composition is converted an 8-membered ringisoprene dimer comprising dimethylcyclooctadienes such as1,5-dimethylcycloocta-1,5-diene and 1,6-dimethylcycloocta-1,5-diene. Insome embodiments, the catalyst system comprises a nickel compound.Ni-catalyzed dimerization of isoprene yields adimethyl-1,5-cyclooctadiene mixture consisting of about 90 to 10, 80 to20, 70 to 30, 60 to 40, 50 to 50, 40 to 60, 30 to 70, 20 to 80 and 10 to90 ratio of 1,5-dimethyl-1,5-cyclooctadiene to1,6-dimethyl-1,5-cyclooctadiene. In one embodiment, the catalyst systemcomprises a 3-component catalyst containing Ni carboxylates orβ-ketones, organoaluminum or organomagnesium compounds and substitutedtriphenylphosphite. For example, a degassed solution of a highly pureisoprene starting composition in anhydrous toluene is mixed withNi(acac)₂, Et₃Al, and tris(3,4-bis(dimethylamino)phenyl)phosphite undernitrogen atmosphere and the mixture is heated at 95° C. to givedimethylcyclooctadiene. In another embodiment, the catalyst systemcomprises Fe carboxylates or β-diketone compounds, organo-Al or Mgcompounds, and 2,2′-dipyridyl derivatives having electron-donatinggroups. For example, a starting isoprene composition is heated with amixture of Fe acetylacetonate, 4,4′-dimethyl-2,2′-dipyridyl, and Et₃Alin toluene to gave 1,6-dimethyl-1,5-cyclooctadiene in good yields. Insome preferred embodiments, the starting isoprene composition is abioisoprene composition.

In some embodiments, the method for producing a fuel constituent fromisoprene comprises contacting the highly pure isoprene composition witha catalyst system to convert at least a portion of the starting isoprenecomposition to unsaturated isoprene dimers and/or trimers. In someembodiments, the catalyst system comprises a Ru, Fe, Ni or Pd catalystor a combination thereof and the isoprene in the starting composition isconverted to a mixture comprising 6-membered ring isoprene dimers. Inone embodiment, the catalyst system is aFe(NO)₂C1-bis(1,5-cyclooctadiene)nickel catalyst system and the isoprenein the starting composition is cyclodimerized at low temperatures (e.g.from −5 to +20° C.) to give 1-methyl- and2-methyl-4-isopropenyl-1-cyclohexene and 1,4- and2,4-dimethyl-4-vinyl-1-cyclohexene. In another embodiment, contactingwith a mixed catalyst of a tris(substituted hydrocarbyl) phosphite,arsenite, or antimonite and a Group VIII metal(0) compound (e.g. Niacetylacetonate) converts the isoprene in the starting composition to1,4-dimethyl-4-vinyl-1-cyclohexene and 1,5-dimethyl-1,5-cyclooctadiene.In another embodiment, contacting an isoprene starting composition witha ruthenium catalyst (e.g. see Itoh, Kenji; Masuda, Katsuyuki; Fukahori,Takahiko; Nakano, Katsumasa; Aoki, Katsuyuki; Nagashima, Hideo,Organometallics (1994), 13(3), 1020-9) converts isoprene to C10 cyclicdimers (e.g. a mixture of dimethyl-cyclooctadienes). The rutheniumcatalyst can be synthesized in two steps from RuCl₃ andpentamethylcyclopentadiene (C₅Me₅H). The process may be performed inbatch mode and the catalyst recovered and recycled. In some preferredembodiments, the starting isoprene composition is a bioisoprenecomposition. One embodiment of the method is illustrated in Scheme XI.

In some embodiments, the method for producing a fuel constituent fromisoprene comprises contacting the highly pure isoprene composition witha catalyst system to convert at least a portion of the starting isoprenecomposition to unsaturated isoprene dimers and/or trimers. In someembodiments, the catalyst system comprises a Ni, Ti, Al, or Mg catalystor a mixture thereof and the isoprene in the starting composition isconverted to a mixture comprising cyclic isoprene trimers such astrimethylcyclododecatrienes. In one embodiment, the catalyst system forthe trimerization of isoprene comprising Ni and/or Ti, one or moreorganometallic compound, and a Group VA compound. The reaction may beconducted in a hydroxyl group-containing solvent. In another embodiment,oligomerization of isoprene catalyzed by nickel naphthenate andisoprenemagnesium in the presence of various phosphites as electrondonors give cyclic dimers containing dimethylcyclooctadiene, inparticular 1,1,1-tris(hydroxymethyl)propane phosphite givestrimethylcyclododecatriene selectively.

In some embodiments, the method for producing a fuel constituent fromisoprene comprises contacting the highly pure isoprene composition witha catalyst system to convert at least a portion of the starting isoprenecomposition to unsaturated isoprene dimers and/or trimers. In someembodiments, the catalyst system comprises a Ni, Pd or Cr catalyst or amixture thereof and the isoprene in the starting composition isconverted telomers or linear dimers and/or trimers. In one embodiment, acatalyst system comprising Pd(0) complexes (e.g. Pd(acac)₂-Ph₃P andPd(OAc)₂-Ph₃P) catalyzes dimerization and telomerization of isoprene togive linear isoprene dimers (e.g. 2,7-dimethyl-1,3,7-octatriene). In onevariation, the reaction is performed in methanol as a solvent and methylethers such as methoxydimethyloctadienes (e.g.1-methoxy-2,7-dimethyl-2,7-octadiene and3-methoxy-2,7-dimethyl-1,7-octadiene) are produced. In anotherembodiment, the catalyst system comprises divalent and trivalenttransition metal-exchanged montmorillonites (e.g. Cr³⁺-montmorillonite).In another embodiment, the catalyst system comprises aNi(0)-aminophosphinite catalyst and the isoprene in the startingcomposition is converted to regioselective tail-to-tail linear dimers.In some variations, the linear dimer formation is accompanied by acompetitive cyclodimerization reaction. In another embodiment, thecatalyst system comprises a chromium N,N-bis(diarylphosphino)aminecatalyst (e.g. see Bowen, L.; Charernsuk, M.; Wass, D. F. Chem. Commun.(2007) 2835-2837) and the isoprene is converted to linear and cyclic C15trimers. In one variation, the chromium catalyst is made in situ fromCrCl₃(THF)₃ and a PNP phosphine ligand.

In some embodiments, the method for producing a fuel constituent from abioisoprene composition comprises chemically transforming a substantialportion of the isoprene in the bioisoprene composition by (i) contactingthe bioisoprene composition with a catalyst for catalyzing trimerizationof isoprene to produce an unsaturated isoprene trimer and (ii)hydrogenating the isoprene trimer to produce a fuel constituent. In someembodiments, at least about 95% of isoprene in the bioisoprenecomposition is converted to non-isoprene compounds during the chemicaltransformation.

In some embodiments, the method for producing a fuel constituent fromisoprene comprises: (a) obtaining a commercially beneficial amount ofany of the highly pure isoprene starting composition described herein;(b) converting at least a portion of the starting isoprene compositionto unsaturated isoprene derivatives; and (c) hydrogenating theunsaturated isoprene derivatives to produce saturated compounds. In someembodiments, hydrogenation of unsaturated isoprene derivatives tosaturated compounds is performed in batch mode. In some embodiments,hydrogenation of unsaturated isoprene derivatives to saturated compoundsis performed in continuous mode. One embodiment of the method isillustrated in Scheme XII.

In some embodiments, the method for producing a fuel constituent fromisoprene comprises: (a) obtaining a commercially beneficial amount ofany of the highly pure isoprene starting composition described herein;(b) converting at least a portion of the starting isoprene compositionto oxygenated isoprene derivatives; and optionally (c) hydrogenating anyunsaturated oxygenated isoprene derivatives to produce saturatedoxygenates. In some embodiments, the isoprene starting composition iscontacted with a catalyst in the presence of alcohols (e.g. methanol orethanol or mixtures thereof) to form oxygenated isoprene derivativescomprise one or more unsaturated or saturated ethers (e.g. methyl ethersor ethyl ethers or mixtures thereof). In some embodiments, the catalystis an acid catalyst such as sulfuric acid, solid phase sulfuric acid(e.g., Dowex Marathon®), liquid and solid-phase fluorosulfonic acids(e.g. trifluoromethanesulfonic acid and Nafion-H (DuPont)). Zeolitecatalysts can also been used, for example beta-zeolite, under conditionssimilar to those described by Hensel et al. [Hensen, K.; Mahaim, C.;Holderich, W. F., Applied Catalysis A: General (1997) 140(2), 311-329.]for the methoxylation of limonene and related monoterpenes. In someembodiments, the isoprene starting compositions or the unsaturatedisoprene dimers or trimers undergo oxidation/hydrogenation to form thehydroxylated isoprene derivatives comprise one or more alcohols such asC5, C10 and C15 alcohols and diols. In some embodiments, the isoprenestarting composition undergoes hydroxylation/esterification to formalcohols and esters. For example, bioisoprene or an unsaturatedintermediate undergoes peroxidation to epoxides with peracids such asperacetic and 3-chloroperbenzoic acid; and hydration to give alcoholsand diols with i) water and acid catalysts and ii) hydroborationmethods. Such reactions are described in, for example, Michael B. Smithand Jerry March, Advanced Organic Chemistry: Reactions, Mechanisms, andStructure, Sixth Edition, John Wiley & Sons, 2007. In some preferredembodiments, the starting isoprene composition is a bioisoprenecomposition.

In some embodiments, the method for producing a fuel constituent fromisoprene comprises: (a) obtaining a commercially beneficial amount ofany of the highly pure isoprene starting composition described herein;(b) partially hydrogenating the starting isoprene composition tomono-olefins (e.g. 2-methylbut-1-ene, 3-methyl-but-1-ene and2-methylbut-2-ene); and (c) producing the fuel constituent from themono-olefins. In one embodiment, the mono-olefin undergoes dimerization.In another embodiment, the mono-olefin is reacted with other olefinsusing traditional hydrocarbon cationic catalysis, such as that used tocovert isobutylene to isooctane. The product of dimerization or reactionwith other olefins are then undergoes hydrogenation to give saturatedfuel constituents. In some preferred embodiments, the highly pureisoprene is a bioisoprene composition. In some embodiment, the startingisoprene composition partially hydrogenated using a palladium catalyst,such as Pd/CaCO₃, Pd/BaSO₄, Pd/C, Pd black, Pd/SiO₃, Pd/Al₂O₃, orPd/SiO₂. In some embodiment, the starting isoprene composition partiallyhydrogenated using a silica-supported polyamidoamine (PAMAM)dendrimer-palladium complex. In some embodiment, the starting isoprenecomposition partially hydrogenated using a palladium-gold andpalladium-silver catalysts (e.g., Pd—Au/SiO₂ and Pd—Ag/SiO₂) have highselectivity for reducing isoprene to mono-olefins over fully reduced C5alkanes. In some embodiment, the starting isoprene composition partiallyhydrogenated using a Group VIB metal on an inorganic support (e.g., ametal zeolite) as a catalyst, e.g. a Mo/Al₂O₃ catalyst. In someembodiment, the starting isoprene composition partially hydrogenatedusing a monolithic catalyst bed, which may be in a honeycombconfiguration. Catalyst support materials for the monolithic catalystbed may include metals such as nickel, platinum, palladium, rhodium,ruthenium, silver, iron, copper, cobalt, chromium, iridium, tin, andalloys or mixtures thereof. In some embodiment, the starting isoprenecomposition partially hydrogenated using an eggshell Pd/d-Al₂O₃catalyst, particularly if the reaction is free of water. In someembodiment, the starting isoprene composition partially hydrogenatedusing a Group VIII metal catalyst promoted by a metal from Group IB,VIIB, VIIB, or zinc to reduce poisoning of the catalyst. Methods forconverting isoprene into isoamylenes (mono-olefins) can be performed incontinuous or batch mode using both homogeneous or heterogeneouscatalysts. One embodiment of the method is illustrated in Scheme XIII.

In some embodiments, isoamylenes produce from partial hydrogenatin ofisoprene undergos trimerization in liquid phase over ion exchange resinsand zeolites. See, GranGranollers, Ind. Eng. Chem. Res., 49 (8), pp3561-3570 (2001).

In some embodiments, the method for producing a fuel constituent fromisoprene comprises: (a) obtaining a commercially beneficial amount ofany of the highly pure isoprene starting composition described herein;(b) partially hydrogenating the starting isoprene composition to amono-olefin (e.g. 2-methylbut-1-ene, 3-methyl-but-1-ene and2-methylbut-2-ene); (c) contacting the mono-olefin with a catalyst fordimerization of mono-olefins to form a dimate with another mono-olefin;and (d) hydrogenating the product of dimerization (dimate) to producesaturated hydrocarbon fuel constituents. In some preferred embodiments,the highly pure isoprene is a bioisoprene composition. In someembodiments, the other mono-olefin is an isoamylene derived from thehighly pure isoprene starting composition. In some embodiments, theother mono-olefin is an olefin from another source such as propylene,butane or isobutene. In some embodiments, the catalyst for dimerizationis a resin catalyst (e.g., Amberlyst, Amberlyst 35, Amberlyst 15,Amberlyst XN1010, Amberlyst XE586). In some embodiments, the catalystfor dimerization is a catalytic material containing an acidic mesoporousmolecular sieve, such as a mesoporous sieve embedded in a zeolitestructure, ZSM-5, ferrierite, ZSM-22, or ZSM-23. In some embodiments,the catalyst for dimerization is the catalytic material is thermallystable at high temperatures (e.g., at least 900° C.). In someembodiments, the catalyst for dimerization is a catalytic materialcontaining an acidic mesoporous molecular sieve. In some embodiments,the catalyst for dimerization is a solid acidic catalyst (e.g., a solidphosphoric acid catalyst, acidic ion exchange resins). One embodiment ofthe method is illustrated in Scheme XIV.

In some embodiments, the method for producing a fuel constituent fromisoprene comprises: (a) obtaining a commercially beneficial amount ofany of the highly pure isoprene starting composition described herein;(b) partially hydrogenating the starting isoprene composition to anisoamylene (e.g. 2-methylbut-1-ene, 3-methyl-but-1-ene and2-methylbut-2-ene); (c) contacting the isoamylene with an alcohol and acatalyst to form an oxygenate; and (c) producing producing the fuelconstituent from the isoamylene oxygenate. In some preferredembodiments, the highly pure isoprene is a bioisoprene composition. Insome embodiments, the isoamylene is contacted with ethanol and the fueloxygenate formed is an ether such as TAME.

In some embodiments, any of the methods described herein further includepurifying the starting isoprene composition before the chemicaltransformation. For example, the isoprene produced using thecompositions and methods of the invention can be purified using standardtechniques. Purification refers to a process through which isoprene isseparated from one or more components that are present when the isopreneis produced. In some embodiments, the isoprene is obtained as asubstantially pure liquid. Examples of purification methods include (i)distillation from a solution in a liquid extractant and (ii)chromatography. As used herein, “purified isoprene” means isoprene thathas been separated from one or more components that are present when theisoprene is produced. In some embodiments, the isoprene is at leastabout 20%, by weight, free from other components that are present whenthe isoprene is produced. In various embodiments, the isoprene is atleast or about 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, or 99%,by weight, pure. Purity can be assayed by any appropriate method, e.g.,by column chromatography, HPLC analysis, or GC-MS analysis. In someembodiments, bioisoprene is recovered from fermentation off-gas byinitial adsorption to activated carbon, followed by desorption andcondensation to give a liquid isoprene composition for chemicaltransformation to a fuel constituent.

Continuous Process for Producing Fuels

The invention also provides a continuous process for producing a fuelconstituent from isoprene comprising: (a) continuously producing acommercially beneficial amount of highly pure isoprene; and (b)continuously transforming chemically at least a portion of thecommercially beneficial amount of highly pure isoprene to a fuelconstituent. In a continuous process according to the invention, astream of highly pure isoprene is produced continuously, for example, bya biological process (e.g. culturing cells producing isoprene) and ispassed to a reactor for chemical transformation. In some embodiments,the isoprene from a fermentation off-gas is separated from water andother permanent gases (e.g., O₂, N₂ and CO₂) using extractivedistillation, condensation, adsorption or membranes in a continuouspurification unit, where the oxygen level is lowered to ppm range andother impurities of concern are removed; the isoprene vapor iscatalytically converted to dimers (C10) and trimers (C15) using aheterogeneous catalyst; the desired product is separated and theunreacted isoprene is returned to the reactor for further conversion. Insome embodiments, the catalytic dimerization is monitored by measuringthe levels of desirable C10 hydrocarbons in the gas phase.

In some embodiments, a stream of biologically produced isoprene ispassed through a chemical reactor (e.g., a reaction tube) maintained ata temperature of about 100° C. to about 300° C., where the biologicallyproduced isoprene undergoes dimerization reactions. In one variation,the isoprene dimers are separated from the product stream and at least aportion of the unreacted isoprene is recycled back into the chemicalreactor. In one embodiment, the unsaturated isoprene dimers produced iscontacted with a hydrogenation catalyst and a hydrogen source in asecond chemical reactor producing partially hydrogenated and/or fullyhydrogenated products. The process can further comprise a step ofrecovering isoprene from the off-gas of a bio-reactor producingisoprene.

In some embodiments, a stream of bioisoprene containing co-producedhydrogen is first separated from the hydrogen gas, the bioisoprenestream is passed through a chemical reactor (e.g., a reaction tube)maintained at a temperature of about 100° C. to about 300° C., where thebiologically produced isoprene undergoes dimerization reactions. In onevariation, the isoprene dimers are separated from the product stream andat least a portion of the unreacted isoprene is recycled back into thechemical reactor. In one embodiment, the unsaturated isoprene dimersproduced is contacted with a hydrogenation catalyst and a hydrogensource in a second chemical reactor producing partially hydrogenatedand/or fully hydrogenated products. In one variation, the hydrogenstream isolated from the isoprene-hydrogen co-production is used in thehydrogenation step. The process can further comprise a step ofrecovering isoprene from the off-gas of a bio-reactor producingisoprene. The process can further comprise a step of purifying thehydrogen stream isolated from the isoprene-hydrogen co-production usingpurification methods known in the art such as cryogenic condensation andsolid matrix adsorption.

Removal of Dienes and Polymers from Fuel Products

Fuel compositions often contain unsaturated compounds (olefins,diolefins and polyolefins) that can form gums, resins, polymers andother undesirable byproducts over time (for example, see Pereira andPasa (2006) Fuel, 85, 1860-1865 and references therein). In general, asthe degree of unsaturation increases of a given compound, the morelikely that compound is to form such byproducts. Isoprene is a 1,3-dienethat readily forms undesirable polymeric byproducts when present in fuelcompositions. While there exist fuel additives (anti-oxidants, radicalquenchers etc.) that can reduce the extent of byproduct formation, suchbyproducts can still form over time. Olefins can also contribute to theformation of ground-level ozone when released into the atmosphere uponevaporation from fuels, or as the result of incomplete combustion ofolefin-containing fuels.

Accordingly, fuel compositions derived wholly or in part from isopreneshould contain little to no free isoprene. A range of methods can beused to either remove isoprene from fuel compositions such aspurification by distillation, reaction with alcohols to form ethers, orhydrogenation to convert isoprene to saturated derivatives. Alternately,isoprene can be treated with a dienophile such as malic anhydrideproducing inert adducts that do not contribute to the formation ofundesirable byproducts.

Provided are fuel compositions comprising isoprene derivatives that aresubstantially free of isoprene. In some embodiments, the fuelcomposition contains less than 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 10% or15% isoprene. Also provided are methods for chemically transforming abioisoprene composition to fuel compositions comprising isoprenederivatives that are substantially free of isoprene. In someembodiments, a substantial portion of the isoprene from the bioisoprenecomposition is converted to fuel constituents. In some embodiments, asubstantial portion of the isoprene from the bioisoprene composition isconverted to intermediates that can be further converted to produce fuelconstituents. In some embodiments, a substantial portion of the isoprenefrom the bioisoprene composition is converted to compounds other thanisoprene. A “substantial portion” is 99.99%, 99.9%, 99%, 98%, 97%, 96%,95%, 90%, 85% or 80%.

In some instances, isoprene and other conjugated dienes can formpolymeric products with gum-like consistencies that can reduce theyields of desired products and/or deactivate catalysts. (See, e.g., R.C. C. Pereira and V. M. D. Pasa. Fuel 85 (2006) 1860-1865.) In someembodiments, methods are provided for determining the amount ofconjugated diene present in a product mixture. D. F. Andrade et al.describe various methods for determining the amount of conjugated dienepresent (Fuel (2010), doi:10.106/j.fuel/2010.01.003), including thefollowing: 1) UOP-326 method (maleic anhydride method), asemi-quantitative method in which the amount of maleic anhydrideconsumed via Diels-Alder reaction with the diene is measured; 2)polarography; 3) gas chromatography, in which the diene may be reactedwith a derivatization agent, such as 4-methyl-1,2,4-triazoline-3,5-dione(MTAD) or 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD); 4) HPLC; 5)supercritical fluid chromatography; 6) NMR; 7) UV and near IRspectroscopy; and 8) other spectroscopic methods, which may includefirst derivatizing the diene with p-nitrobenezenediazonium fluoroborate.

Method are provided herein for minimizing gum-formation and/or reducingthe amount of gum that has been produced. In some embodiments, ananti-oxidant is used to minimize gum-formation. In other embodiments,polymeric by-products are recycled back to the process stream. In someembodiments, depolymerization of polymeric by-products is carried outvia olefin metathesis. Olefin metathesis can be carried out using amolybdenum or tungsten catalyst, such as 2,6-diisopropylphenylimidoneophylidene molybdenum bis(2-tertbutylphenoxide). (See U.S. Pat. No.5,446,102.)

In some embodiments, any of the methods described herein further includecharacterizing the products of these reactions and assessing thepotential fuel value. For example, the products are characterized bystandard methods known in the art, e.g. GC/MS, NMR, UV-Vis and IRspectroscopies, boiling temperature, density and other physicalproperties. The products can be further characterized by dualcarbon-isotopic fingerprinting (see, U.S. Pat. No. 7,169,588). Thepotential fuel value of the products are assessed by parametersmeasuring fuel properties such as the energy density, heating value,water solubility, octane/cetane number, density, viscosity, surfacetension, enthalpy of vaporization, vapor diffusivity, flash point,autoignition point, flammability limits, cloud point and chemicalstability.

As with commercial petroleum fuels, BioIsoFuel™ products can be testedfor their acidity, density, trace mineral content, benzene, totalaromatics content, water content, and corrosivity. To assure that minorimpurities in BioIsoFuel™ products are not adversely affecting theirmaterial properties, samples can also be tested for their corrosivityand compatibility with fuel systems by standard ASTM tests such as KarlFischer for water and copper strip for corrosivity.

Fuel Compositions

The invention provides a fuel composition comprising a fuel constituentproduced by any of the methods and processes described herein. In someembodiments, the fuel constituent comprises one or more of thehydrocarbons selected from the group consisting of saturated C10 andC₁₋₅ hydrocarbons derived from isoprene and oxygenated derivatives ofisoprene. The hydrocarbon BioIsoFuel™ products are expected to becompletely compatible with petroleum gasoline and diesel. Blends ofBioIsoFuel™ with commercial petroleum fuels are expected to have moredesirable properties, as they do not contain acids, sulfur, aromatics,or other undesirable impurities.

In some preferred embodiments, the fuel constituent comprisesderivatives of bioisoprene. It is anticipated that there will be asignificant demand for fuels derived from bioisoprene which is made fromrenewable, non-petrochemical based resources. It is believed thatindustrial customers and consumers would prefer to purchase fuelcomponents derived from such environmentally friendly sources to thosethat are made with isoprene derived from a petrochemical process. It isfurther believed that customers would be willing to pay premium pricesfor such environmentally friendly products that are made with renewableresources. Fuel components derived from bioisoprene compositionsdescribed herein offer the benefit of being verifiable as to be derivedfrom non-petrochemical based resources.

In some embodiments, the fuel constituent comprises one or more of thesaturated cyclic C10 hydrocarbons such as 1,2-bis(isopropyl)cyclobutane,1,2-bis(isopropyl)cyclobutane, 1-methyl-4-isopropylcyclohexane,1-methyl-3-isopropylcyclohexane, 1-ethyl-1,4-dimethylcyclohexane,1-ethyl-1,3-dimethylcyclohexane, 1,5-dimethylcyclooctane and1,4-dimethylcyclooctane. In some embodiments, the fuel constituentcomprises one or more of the saturated cyclic C₁₋₅ hydrocarbons such as1,5,9-trimethylcyclododecane and 1,5,10-trimethylcyclododecane. In someembodiments, the fuel constituent comprises one or more of the saturatedaliphatic C10 hydrocarbons such as 2,6-dimethyloctane,2,7-dimethyloctane and 3,6-dimethyloctane. In some embodiments, the fuelconstituent comprises one or more of the saturated aliphatic C15hydrocarbons such as 2,6,10-trimethyldodecane, 2,7,10-trimethyldodecaneand 3,7,10-trimethyldodecane. In some embodiments, the fuel constituentcomprises 2-methylbutane. In some embodiments, the fuel constituentcomprises one or more of the hydrocarbons selected from the groupconsisting of 1,2-bis(isopropyl)cyclobutane,1,2-bis(isopropyl)cyclobutane, 1-methyl-4-isopropylcyclohexane,1-methyl-3-isopropylcyclohexane, 1-ethyl-1,4-dimethylcyclohexane,1-ethyl-1,3-dimethylcyclohexane, 1,5-dimethylcyclooctane,1,4-dimethylcyclooctane, 1,5,9-trimethylcyclododecane,1,5,10-trimethylcyclododecane, 2,6-dimethyloctane, 2,7-dimethyloctane,3,6-dimethyloctane, 2,6,10-trimethyldodecane, 2,7,10-trimethyldodecane,3,7,10-trimethyldodecane and 2-methylbutane.

In some embodiments, the fuel constituent comprises one or more of thesaturated hydroxylates such as C5 alcohols and diols (e.g.3-methyl-butan-1-ol, 2-methyl-butan-1-ol and 2-methyl-butan-2-ol,3-methyl-butan-1,3-diol and 2-methyl-butan-2,3-diol), C-10 alcohols anddiols (e.g. 3,7-dimethyloctan-1-ol, 2,7-dimethyloctan-1-ol,2,7-dimethyloctan-2-ol and 2,7-dimethyloctan-2,7-diol) and cyclic C-10alcohols (e.g. 2-(4-methylcyclohexyl)propan-2-ol,2-(4-methylcyclohexyl)propan-1-ol, 2-(1,4-dimethylcyclohexyl)ethanol and4-ethyl-1,4-dimethylcyclohexanol). In some embodiments, the fuelconstituent comprises one or more of the saturated ethers such as1,3-diethoxy-3-methylbutane, 1-ethoxy-3-methylbutane,1-methoxy-2,7-dimethyloctane and 3-methoxy-2,7-dimethyloctane. In someembodiments, the fuel constituent comprises one or more of the isoprenederived oxygenates selected from the group consisting of3-methyl-butan-1-ol, 2-methyl-butan-1-ol, 2-methyl-butan-2-ol,3-methyl-butan-1,3-diol, 2-methyl-butan-2,3-diol,3,7-dimethyloctan-1-ol, 2,7-dimethyloctan-1-ol, 2,7-dimethyloctan-2-ol,2,7-dimethyloctan-2,7-diol, 2-(4-methylcyclohexyl)propan-2-ol,2-(4-methylcyclohexyl)propan-1-ol, 2-(1,4-dimethylcyclohexyl)ethanol,4-ethyl-1,4-dimethylcyclohexanol, 1,3-diethoxy-3-methylbutane,1-ethoxy-3-methylbutane, 1-methoxy-2,7-dimethyloctane and3-methoxy-2,7-dimethyloctane.

In some embodiments, the fuel constituent comprises less than or about0.5 μg/L a product from a C5 hydrocarbon other than isoprene afterundergoing the steps according to any of the methods and processesdescribed herein. In one embodiment, the fuel constituent issubstantially free of a product from a C5 hydrocarbon other thanisoprene after undergoing the steps according to any of the methods andprocesses described herein. In some embodiments, the fuel constituentcomprises less than or about 0.2, 0.12, 0.10, 0.08, 0.06, 0.04, 0.02,0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001% C5 hydrocarbonsother than isoprene (such 1,3-cyclopentadiene, cis-1,3-pentadiene,trans-1,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene,2-methyl-1-butene, 3-methyl-1-butyne, pent-4-ene-1-yne,trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne) by weight compared tothe total weight of all C5 hydrocarbons in the starting composition. Insome embodiments, the fuel constituent comprises C5 hydrocarbons otherthan isoprene (such 1,3-cyclopentadiene, trans-1,3-pentadiene,cis-1,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene,2-methyl-1-butene, 3-methyl-1-butyne, pent-4-ene-1-yne,trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne) at a concentration ofless than 100, 10, 1, 0.1 or 0.01 ppm.

It is understood that components other than isoprene in the bioisoprenecompositions described herein will be converted to different productsafter undergoing various processes described herein. The sensitivitiesof metal-based catalysts used in the methods/processes to the componentsother than isoprene are different depending on the nature and levels ofthese components. For the thermal dimerization process, a far widerrange of compounds will be tolerated. In some cases, a component otherthan isoprene will react with isoprene to produce adducts, for examplethe Diels-Alder reactions of methacrolein and methyl vinyl ketone withisoprene to give 6-membered products. The unsaturated adducts can befurther hydrogenated to saturated derivatives which are present in thefuel constituents and compositions, e.g.1,4-dimethyl-1-(hydroxymethyl)cyclohexane and1-(1-hydroxyethyl)-4-methylcyclohexane (see Scheme XV).

In some embodiments, the fuel constituent comprises a product from acompound selected from the group consisting of ethanol, acetone, C5prenyl alcohols, and isoprenoid compounds with 10 or more carbon atomsafter undergoing the steps according to any of the methods and processesdescribed herein. In some embodiments, the fuel constituent comprisesone or more products from one or more compounds selected from the groupconsisting of ethanol, acetone, methanol, acetaldehyde, methacrolein,methyl vinyl ketone, 2-methyl-2-vinyloxirane, cis- andtrans-3-methyl-1,3-pentadiene, a C5 prenyl alcohol (such as3-methyl-3-buten-1-ol or 3-methyl-2-buten-1-ol), 2-heptanone,6-methyl-5-hepten-2-one, 2,4,5-trimethylpyridine,2,3,5-trimethylpyrazine, citronellal, methanethiol, methyl acetate,1-propanol, diacetyl, 2-butanone, 2-methyl-3-buten-2-ol, ethyl acetate,2-methyl-1-propanol, 3-methyl-1-butanal, 3-methyl-2-butanone, 1-butanol,2-pentanone, 3-methyl-1-butanol, ethyl isobutyrate, 3-methyl-2-butenal,butyl acetate, 3-methylbutyl acetate, 3-methyl-3-buten-1-yl acetate,3-methyl-2-buten-1-yl acetate, 3-hexen-1-ol, 3-hexen-1-yl acetate,limonene, geraniol (trans-3,7-dimethyl-2,6-octadien-1-ol), citronellol(3,7-dimethyl-6-octen-1-ol), (E)-3,7-dimethyl-1,3,6-octatriene,(Z)-3,7-dimethyl-1,3,6-octatriene, and 2,3-cycloheptenolpyridine afterundergoing the steps according to any of the methods and processesdescribed herein. In some embodiments, the fuel constituent comprisesone or more compounds selected from the group consisting of1,4-dimethyl-1-(hydroxymethyl)cyclohexane,1-(1-hydroxyethyl)-4-methylcyclohexane, 2-methylbutane, 3-methylpentane,2-propanol, 2-methyl-1-propanol, 2-butanol, 3-methyl-1-butanol, ethylacetate and 3-methyl-1-butyl acetate. In some embodiments, the fuelconstituent comprises one or more compounds selected from the groupconsisting of 1,4-dimethyl-1-(hydroxymethyl)cyclohexane,1-(1-hydroxyethyl)-4-methylcyclohexane, 2-methylbutane, 3-methylpentane,2-propanol, 2-methyl-1-propanol, 2-butanol, 3-methyl-1-butanol, ethylacetate and 3-methyl-1-butyl acetate at levels greater than or about 10ppm, 1 ppm, 100 ppb, 10 ppb or 1 ppb.

In some embodiments, the fuel constituent comprises one or morecompounds selected from the group consisting of ethanol, acetone,methanol, acetaldehyde, methacrolein, methyl vinyl ketone,2-methyl-2-vinyloxirane, cis- and trans-3-methyl-1,3-pentadiene, a C5prenyl alcohol (such as 3-methyl-3-buten-1-ol or 3-methyl-2-buten-1-ol),2-heptanone, 6-methyl-5-hepten-2-one, 2,4,5-trimethylpyridine,2,3,5-trimethylpyrazine, citronellal, methanethiol, methyl acetate,1-propanol, diacetyl, 2-butanone, 2-methyl-3-buten-2-ol, ethyl acetate,2-methyl-1-propanol, 3-methyl-1-butanal, 3-methyl-2-butanone, 1-butanol,2-pentanone, 3-methyl-1-butanol, ethyl isobutyrate, 3-methyl-2-butenal,butyl acetate, 3-methylbutyl acetate, 3-methyl-3-buten-1-yl acetate,3-methyl-2-buten-1-yl acetate, 3-hexen-1-ol, 3-hexen-1-yl acetate,limonene, geraniol(trans-3,7-dimethyl-2,6-octadien-1-ol), citronellol(3,7-dimethyl-6-octen-1-ol), (E)-3,7-dimethyl-1,3,6-octatriene,(Z)-3,7-dimethyl-1,3,6-octatriene, and 2,3-cycloheptenolpyridine atlevels greater than or about 10 ppm, 1 ppm, 100 ppb, 10 ppb or 1 ppb.

Carbon Fingerprinting

BioIsoFuels derived from bioisoprene can be distinguished from fuelsderived form petrochemical carbon on the basis of dual carbon-isotopicfingerprinting. Additionally, the specific source of biosourced carbon(e.g. glucose vs. glycerol) can be determined by dual carbon-isotopicfingerprinting (see, U.S. Pat. No. 7,169,588, which is hereinincorporated by reference).

This method usefully distinguishes chemically-identical materials, andapportions carbon in products by source (and possibly year) of growth ofthe biospheric (plant) component. The isotopes, ¹⁴C and ¹³C, bringcomplementary information to this problem. The radiocarbon datingisotope (¹⁴C), with its nuclear half life of 5730 years, clearly allowsone to apportion specimen carbon between fossil (“dead”) and biospheric(“alive”) feedstocks [Currie, L. A. “Source Apportionment of AtmosphericParticles,” Characterization of Environmental Particles, J. Buffle andH. P. van Leeuwen, Eds., 1 of Vol. I of the IUPAC EnvironmentalAnalytical Chemistry Series (Lewis Publishers, Inc) (1992) 3 74]. Thebasic assumption in radiocarbon dating is that the constancy of ¹⁴Cconcentration in the atmosphere leads to the constancy of ¹⁴C in livingorganisms. When dealing with an isolated sample, the age of a sample canbe deduced approximately by the relationship t=(−5730/0.693)ln(A/A_(O))(Equation 14) where t=age, 5730 years is the half-life of radiocarbon,and A and A_(O) are the specific ¹⁴C activity of the sample and of themodern standard, respectively [Hsieh, Y., Soil Sci. Soc. Am J., 56, 460,(1992)]. However, because of atmospheric nuclear testing since 1950 andthe burning of fossil fuel since 1850, ¹⁴C has acquired a second,geochemical time characteristic. Its concentration in atmosphericCO₂—and hence in the living biosphere—approximately doubled at the peakof nuclear testing, in the mid-1960s. It has since been graduallyreturning to the steady-state cosmogenic (atmospheric) baseline isotoperate (¹⁴C/¹²C) of ca. 1.2×10⁻¹², with an approximate relaxation“half-life” of 7-10 years. (This latter half-life must not be takenliterally; rather, one must use the detailed atmospheric nuclearinput/decay function to trace the variation of atmospheric andbiospheric ¹⁴C since the onset of the nuclear age.) It is this latterbiospheric ¹⁴C time characteristic that holds out the promise of annualdating of recent biospheric carbon. ¹⁴C can be measured by acceleratormass spectrometry (AMS), with results given in units of “fraction ofmodern carbon” (f_(M)). f_(M) is defined by National Institute ofStandards and Technology (NIST) Standard Reference Materials (SRMs)4990B and 4990C, known as oxalic acids standards HOxI and HOxII,respectively. The fundamental definition relates to 0.95 times the¹⁴C/¹²C isotope ratio HOxI (referenced to AD 1950). This is roughlyequivalent to decay-corrected pre-Industrial Revolution wood. For thecurrent living biosphere (plant material), f_(M) ^(˜)1.1.

The stable carbon isotope ratio (¹³C/¹²C) a complementary route tosource discrimination and apportionment. The ¹³C/¹²C ratio in a givenbiosourced material is a consequence of the ¹³C/¹²C ratio in atmosphericcarbon dioxide at the time the carbon dioxide is fixed and also reflectsthe precise metabolic pathway. Regional variations also occur.Petroleum, C₃ plants (the broadleaf), C₄ plants (the grasses), andmarine carbonates all show significant differences in ¹³C/¹²C and thecorresponding d¹³C values. Furthermore, lipid matter of C₃ and C₄ plantsanalyze differently than materials derived from the carbohydratecomponents of the same plants as a consequence of the metabolic pathway.Within the precision of measurement, ¹³C shows large variations due toisotopic fractionation effects, the most significant of which for theinstant invention is the photosynthetic mechanism. The major cause ofdifferences in the carbon isotope ratio in plants is closely associatedwith differences in the pathway of photosynthetic carbon metabolism inthe plants, particularly the reaction occurring during the primarycarboxylation, i.e., the initial fixation of atmospheric CO₂. Two largeclasses of vegetation are those that incorporate the “C₃” (orCalvin-Benson) photosynthetic cycle and those that incorporate the “C₄”(or Hatch-Slack) photosynthetic cycle. C₃ plants, such as hardwoods andconifers, are dominant in the temperate climate zones. In C₃ plants, theprimary CO₂ fixation or carboxylation reaction involves the enzymeribulose-1,5-diphosphate carboxylase and the first stable product is a3-carbon compound. C₄ plants, on the other hand, include such plants astropical grasses, corn and sugar cane. In C₄ plants, an additionalcarboxylation reaction involving another enzyme, phosphoenol-pyruvatecarboxylase, is the primary carboxylation reaction. The first stablecarbon compound is a 4-carbon acid which is subsequently decarboxylated.The CO₂ thus released is refixed by the C₃ cycle.

Both C₄ and C₃ plants exhibit a range of ¹³C/¹²C isotopic ratios, buttypical values are ca. −10 to −14 per mil (C₄) and −21 to −26 per mil(C₃₋) [Weber et al., J. Agric. Food Chem., 45, 2942 (1997)]. Coal andpetroleum fall generally in this latter range. The ¹³C measurement scalewas originally defined by a zero set by pee dee belemnite (PDB)limestone, where values are given in parts per thousand deviations fromthis material. The “d¹³C”, values are in parts per thousand (per mil),abbreviated ‰, and are calculated as follows (Equation 15):

$\begin{matrix}{{\delta^{13}C} \equiv {\frac{\left( {}^{13}{C/^{12}C} \right)_{sample} - \left( {}^{13}{C/^{12}C} \right)_{standard}}{\left( {}^{13}{C/^{12}C} \right)_{standard}} \times 100\%}} & \left( {{Equation}\mspace{14mu} 15} \right)\end{matrix}$

Since the PDB reference material (RM) has been exhausted, a series ofalternative RMs have been developed in cooperation with the IAEA, USGS,NIST, and other selected international isotope laboratories. Notationsfor the per mil deviations from PDB is d¹³C. Measurements are made onCO₂ by high precision stable ratio mass spectrometry (IRMS) on molecularions of masses 44, 45 and 46.

For isoprene derived from extractive distillation of C₅ streams frompetroleum refineries, δ¹³C is about −22‰ to about −24‰. This range istypical for light, unsaturated hydrocarbons derived from petroleum, andproducts derived from petroleum-based isoprene typically containisoprenic units with the same δ¹³C. Bioisoprene produced by fermentationof corn-derived glucose (δ¹³C −10.73‰) with minimal amounts of othercarbon-containing nutrients (e.g., yeast extract) produces isoprenewhich can be polymerized into polyisoprene with δ¹³C −14.66‰ to −14.85‰.Products produced from such bioisoprene are expected to have δ¹³C valuesthat are less negative than those derived from petroleum-based isoprene.For isoprene derived from the reaction of isobutylene with formaldehyde,δ¹³C values can be about −34.4‰ because formaldehyde is often derivedfrom feedstocks with much more negative δ¹³C values.

The fuels and fuel constituents of this invention which are made withisoprene from the cell cultures that utilize bio-renewable carbonsources can be identified as such by virtue of their d¹³C value andother fuel characteristics. In some embodiments, the fuel constituentderived from bioisoprene has δ¹³C values of greater (less negative) than−22‰. In some embodiments, the fuel constituent derived from bioisoprenehas δ¹³C values of greater than −20, −18, −16, −14, −12, or −10‰. Insome embodiments, the fuel constituent derived from bioisoprene has aδ¹³C value which is within the range of −22 to −10, −21 to −12, or −20to −14‰. In some embodiments, the fuel constituent derived frombioisoprene has a δ¹³C value which is within the range of −34 to −24,−34 to −25, −33 to −25, −32 to −24, −32 to −25, −31 to −25, −30 to −29,−30.0 to −29.5, −29.5 to −28.5, or −29.0 to −28.5‰.

In some embodiments, the fuel constituent derived from bioisoprenecomprises radioactive carbon-14. In some embodiments, the ¹⁴C/¹²C ratiois greater than or about 1.0×10⁻¹², 1.05×10⁻¹², 1.1×10⁻¹², 1.15×10⁻¹²,or 1.2×10⁻¹². In some embodiments, the fuel constituent derived frombioisoprene has an f_(M) value of greater than or about 0.9, 0.95, 1.0,1.05 or 1.1. In some embodiments, the fuel constituent derived frombioisoprene has an f_(M) value of greater than or about 0.9, 0.95, 1.0,1.05 or 1.1 and δ¹³C values of greater (less negative) than −22‰. Insome embodiments, the fuel constituent derived from bioisoprene has anf_(M) value of greater than or about 0.9, 0.95, 1.0, 1.05 or 1.1 and aδ¹³C value which is within the range of −22 to −10, −21 to −12, or −20to −14‰. In some embodiments, the fuel constituent derived frombioisoprene has an f_(M) value of greater than or about 0.9, 0.95, 1.0,1.05 or 1.1 and a δ¹³C value which is within the range of −34 to −24,−34 to −25, −33 to −25, −32 to −24, −32 to −25, −31 to −25, −30 to −29,−30.0 to −29.5, −29.5 to −28.5, or −29.0 to −28.5‰.

The bioisoprene derivatives and the associated BioIsoFuels,intermediates, and mixtures may be completely distinguished from theirpetrochemical derived counterparts on the basis of ¹⁴C (f_(M)) and dualcarbon-isotopic fingerprinting, indicating new compositions of matter.

In some embodiments, the fuel constituent of the invention has an energydensity higher than that of ethanol. In some embodiments, the fuelconstituent boosts the cetane number of a fuel, e.g., a petroleum-basedfuel. In some embodiments, the fuel constituent reduces emission ofpetroleum based fuels. In some embodiments, the fuel composition has anoctane number in the range between about 80 to about 120. In someembodiments, the fuel composition has a cetane number in the rangebetween about 30 to about 130.

In some embodiments, a fuel composition of the invention comprises oneor more dimethylcyclooctane compounds. In some embodiments, a fuelcomposition of the invention comprises one or more C10 hydrocarbons suchas substituted cyclohexanes. In some embodiments, a fuel composition ofthe invention comprises one or more isoprene derived oxygenatesdescribed herein. In some embodiments, any of the fuel compositionsdescribed herein further comprises a petroleum based fuel in the amountof from about 1% to about 95% by weight or volume, based on the totalweight or volume of the total fuel composition.

The invention further provides methods for making a fuel compositioncomprising obtaining a petroleum distillate and adding a fuelconstituent of the invention.

The invention can be further understood by reference to the followingexamples, which are provided by way of illustration and are not meant tobe limiting.

EXAMPLES Example 1 Production of Isoprene in E. coli ExpressingRecombinant Kudzu Isoprene Synthase

I. Construction of Vectors for Expression of the Kudzu Isoprene Synthasein E. coli

The protein sequence for the kudzu (Pueraria montana) isoprene synthasegene (IspS) was obtained from GenBank (AAQ84170). A kudzu isoprenesynthase gene, optimized for E. coli codon usage, was purchased fromDNA2.0 (SEQ ID NO:1). The isoprene synthase gene was removed from thesupplied plasmid by restriction endonuclease digestion with BspLU1IPstI, gel-purified, and ligated into pTrcHis2B (Invitrogen) that hadbeen digested with NcoI/PstI. The construct was designed such that thestop codon in the isoprene synthase gene 5′ to the PstI site. As aresult, when the construct was expressed the His-Tag is not attached tothe isoprene synthase protein. The resulting plasmid, pTrcKudzu, wasverified by sequencing (FIGS. 2 and 3; SEQ ID NO:2).

The isoprene synthase gene was also cloned into pET16b (Novagen). Inthis case, the isoprene synthase gene was inserted into pET16b such thatthe recombinant isoprene synthase protein contained the N-terminal Histag. The isoprene synthase gene was amplified from pTrcKudzu by PCRusing the primer set pET-His-Kudzu-2F:5′-CGTGAGATCATATGTGTGCGACCTCTTCTCAATTTAC (SEQ ID NO:49) andpET-His-Kudzu-R: 5′-CGGTCGACGGATCCCTGCAGTTAGACATACATCAGCTG (SEQ IDNO:50). These primers added an NdeI site at the 5′-end and a BamH1 siteat the 3′ end of the gene respectively. The plasmid pTrcKudzu, describedabove, was used as template DNA, Herculase polymerase (Stratagene) wasused according to manufacture's directions, and primers were added at aconcentration of 10 pMols. The PCR was carried out in a total volume of25 μl. The PCR product was digested with NdeI/BamH1 and cloned intopET16b digested with the same enzymes. The ligation mix was transformedinto E. coli Top10 (Invitrogen) and the correct clone selected bysequencing. The resulting plasmid, in which the kudzu isoprene synthasegene was expressed from the T7 promoter, was designated pETNHisKudzu(FIGS. 4 and 5; SEQ ID NO:51).

The kudzu isoprene synthase gene was also cloned into the low copynumber plasmid pCL1920. Primers were used to amplify the kudzu isoprenesynthase gene from pTrcKudzu described above. The forward primer added aHindIII site and an E. coli consensus RBS to the 5′ end. The PstIcloning site was already present in pTrcKudzu just 3′ of the stop codonso the reverse primer was constructed such that the final PCR productincludes the PstI site. The sequences of the primers were:HindIII-rbs-Kudzu F: 5′-CATATGAAAGCTTGTATCGATTAAATAAGGAGGAATAAACC (SEQID NO:51) and BamH1-Kudzu R:

5′-CGGTCGACGGATCCCTGCAGTTAGACATACATCAGCTG (SEQ ID NO:50). The PCRproduct was amplified using Herculase polymerase with primers at aconcentration of 10 pmol and with 1 ng of template DNA (pTrcKudzu). Theamplification protocol included 30 cycles of (95° C. for 1 minute, 60°C. for 1 minute, 72° C. for 2 minutes). The product was digested withHindIII and PstI and ligated into pCL 1920 which had also been digestedwith HindIII and PstI. The ligation mix was transformed into E. coliTop10. Several transformants were checked by sequencing. The resultingplasmid was designated pCL-lac-Kudzu (FIGS. 6 and 7; SEQ ID NO:4).

II. Determination of Isoprene Production

For the shake flask cultures, one ml of a culture was transferred fromshake flasks to 20 ml CTC headspace vials (Agilent vial cat#5188 2753;cap cat#5188 2759). The cap was screwed on tightly and the vialsincubated at the equivalent temperature with shaking at 250 rpm. After30 minutes the vials were removed from the incubator and analyzed asdescribed below (see Table 1 for some experimental values from thisassay).

In cases where isoprene production in fermentors was determined, sampleswere taken from the off-gas of the fermentor and analyzed directly asdescribed below (see Table 2 for some experimental values from thisassay).

The analysis was performed using an Agilent 6890 GC/MS system interfacedwith a CTC Analytics (Switzerland) CombiPAL autosampler operating inheadspace mode. An Agilent HP-5MS GC/MS column (30 m×0.25 mm; 0.25 μmfilm thickness) was used for separation of analytes. The sampler was setup to inject 500 μL, of headspace gas. The GC/MS method utilized heliumas the carrier gas at a flow of 1 ml/min. The injection port was held at250° C. with a split ratio of 50:1. The oven temperature was held at 37°C. for the 2 minute duration of the analysis. The Agilent 5793N massselective detector was run in single ion monitoring (SIM) mode on m/z67. The detector was switched off from 1.4 to 1.7 minutes to allow theelution of permanent gases. Under these conditions isoprene(2-methyl-1,3-butadiene) was observed to elute at 1.78 minutes. Acalibration table was used to quantify the absolute amount of isopreneand was found to be linear from 1 μg/L to 70,000 μg/L. The limit ofdetection was estimated to be 50 to 100 ng/L using this method.

III. Production of Isoprene in Shake Flasks Containing E. coli CellsExpressing Recombinant Isoprene Synthase

The vectors described above were introduced to E. coli strain BL21(Novagen) to produce strains BL21/ptrcKudzu, BL21/pCL-lac-Kudzu andBL21/pETHisKudzu. The strains were spread for isolation onto LA (Luriaagar)+carbenicillin (50 μg/ml) and incubated overnight at 37° C. Singlecolonies were inoculated into 250 ml baffled shake flasks containing 20ml Luria Bertani broth (LB) and carbenicillin (100 μg/ml). Cultures weregrown overnight at 20° C. with shaking at 200 rpm. The OD₆₀₀ of theovernight cultures were measured and the cultures were diluted into a250 ml baffled shake flask containing 30 ml MagicMedia(Invitrogen)+carbenicillin (100 μg/ml) to an OD₆₀₀˜0.05. The culture wasincubated at 30° C. with shaking at 200 rpm. When the OD₆₀₀˜0.5-0.8, 400μM IPTG was added and the cells were incubated for a further 6 hours at30° C. with shaking at 200 rpm. At 0, 2, 4 and 6 hours after inductionwith IPTG, 1 ml aliquots of the cultures were collected, the OD₆₀₀ wasdetermined and the amount of isoprene produced was measured as describedabove. Results are shown in FIG. 8.

IV. Production of Isoprene from BL21/ptrcKudzu in 14 Liter Fermentation

Large scale production of isoprene from E. coli containing therecombinant kudzu isoprene synthase gene was determined from a fed-batchculture. The recipe for the fermentation media (TM2) per liter offermentation medium was as follows: K₂HPO₄ 13.6 g, KH₂PO₄ 13.6 g,MgSO₄*7H₂O 2 g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3g, (NH₄)₂SO₄ 3.2 g, yeast extract 5 g, 1000× Modified Trace MetalSolution 1 ml. All of the components were added together and dissolvedin diH₂O. The pH was adjusted to 6.8 with potassium hydroxide (KOH) andq.s. to volume. The final product was filter sterilized with 0.22μfilter (only, do not autoclave). The recipe for 1000× Modified TraceMetal Solution was as follows: Citric Acids*H₂O 40 g, MnSO₄*H₂O 30 g,NaCl 10 g, FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO*7H₂O 1 g, CuSO₄*5H₂O 100mg, H₃BO₃ 100 mg, NaMoO₄*2H₂O 100 mg. Each component was dissolved oneat a time in diH₂O, pH to 3.0 with HCl/NaOH, then q.s. to volume andfilter sterilized with a 0.22μ filter.

This experiment was carried out in 14 L bioreactor to monitor isopreneformation from glucose at the desired fermentation, pH 6.7 andtemperature 34° C. An inoculum of E. coli strain BL21/ptrcKudzu takenfrom a frozen vial was prepared in soytone-yeast extract-glucose medium.After the inoculum grew to OD₅₅₀=0.6, two 600 ml flasks were centrifugedand the contents resuspended in 70 ml supernatant to transfer the cellpellet (70 ml of OD 3.1 material) to the bioreactor. At various timesafter inoculation, samples were removed and the amount of isopreneproduced was determined as described above. Results are shown in FIG. 9.

Example 2 Production of Isoprene in E. coli Expressing RecombinantPoplar Isoprene Synthase

The protein sequence for the poplar (Populus alba×Populus tremula)isoprene synthase (Schnitzler, J-P, et al. (2005) Planta 222:777-786)was obtained from GenBank (CAC35696). A gene, codon optimized for E.coli, was purchased from DNA2.0 (p9796-poplar, FIGS. 30 and 31; SEQ IDNO:14). The isoprene synthase gene was removed from the supplied plasmidby restriction endonuclease digestion with BspLU1 IPstI, gel-purified,and ligated into pTrcHis2B that had been digested with NcoI/PstI. Theconstruct is cloned such that the stop codon in the insert is before thePstI site, which results in a construct in which the His-Tag is notattached to the isoprene synthase protein. The resulting plasmidpTrcPoplar (FIGS. 32 and 33; SEQ ID NO:15), was verified by sequencing.

Example 2B Demonstration of Isoprene Synthase Activity from SeveralPopulus Isoprene Synthases

The following isoprene synthases were examined; Populus alba (Accessionnumber BAD98243; FIGS. 137A and B; SEQ ID NO:30), Populus nigra(Accession number CAL69918; FIGS. 137C and D; SEQ ID NO:31), Populustremuloides (Accession number AAQ16588; FIG. 137 E, F, and G; SEQ IDNOs:32-33), Populus trichocarpa (Accession number ACD70404; FIGS. 137Hand I; SEQ ID NO:34), Populus alba×Populus tremula (Accession numberCAJ29303; FIGS. 137J and K; SEQ ID NO:35), and MCM112-Kudzu.

pET24Kudzu (also referred to as MCM112) was constructed as follows: thekudzu isoprene synthase gene was subcloned into the pET24d vector(Novagen) from the pCR2.1 vector (Invitrogen). The kudzu IspS gene wasamplified from pTrcKudzu template DNA using primers MCM50 5′-GATCATGCATTCGCCCTTAG GAGGTAAAAAAACATGTGTGCGACCTCTTC TCAATTTACT (SEQ ID NO:52); andMCM53 5′-CGGTCGACGGATCCCTGCAG TTAGACATAC ATCAGCTG (SEQ ID NO:50). PCRreactions were carried out using Taq DNA Polymerase (Invitrogen), andthe resulting PCR product was cloned into pCR2.1-TOPO TA cloning vector(Invitrogen), and transformed into E. coli Top10 chemically competentcells (Invitrogen). Transformants were plated on L-agar containingcarbenicillin (50 μg/ml) and incubated overnight at 37° C. Five ml LuriaBroth cultures containing carbenicillin 50 μg/ml were inoculated withsingle transformants and grown overnight at 37° C. Five colonies werescreened for the correct insert by sequencing of plasmid DNA isolatedfrom 1 ml of liquid culture (Luria Broth) and purified using the QIAprepSpin Mini-prep Kit (Qiagen). The resulting plasmid, designated MCM93,contains the kudzu IspS coding sequence in a pCR2.1 backbone (FIG.137L). The sequence of MCM93 (SEQ ID NO:36) is shown in FIGS. 137M andN.

The kudzu coding sequence was removed by restriction endonucleasedigestion with PciI and BamH1 (Roche) and gel purified using theQIAquick Gel Extraction kit (Qiagen). The pET24d vector DNA was digestedwith NcoI and BamHI (Roche), treated with shrimp alkaline phosphatase(Roche), and purified using the QIAprep Spin Mini-prep Kit (Qiagen). Thekudzu IspS fragment was ligated to the NcoI/BamH1 digested pET24d usingthe Rapid DNA Ligation Kit (Roche) at a 5:1 fragment to vector ratio ina total volume of 20 μl. A portion of the ligation mixture (5 μl) wastransformed into E. coli Top 10 chemically competent cells and plated onL agar containing kanamycin (50 μg/ml). The correct transformant wasconfirmed by sequencing and transformed into chemically competentBL21(λDE3)pLysS cells (Novagen). A single colony was selected afterovernight growth at 37° C. on L agar containing kanamycin (50 μg/ml). Amap of the resulting plasmid designated as pET24D-Kudzu is shown in FIG.137O. The sequence of pET24D-Kudzu (SEQ ID NO:37) is shown in FIGS. 137Pand Q.

Escherichia coli optimized isoprene synthase genes cloned into thepET24a expression vector (Novagen) were purchased from DNA2.0 (MenloPark, Calif.) for Populus tremuloides, Populus alba, Populus nigra andPopulus trichocarpa. Genes were synthesized with the chloroplast transitpeptide sequence removed, resulting in expression of mature proteins.

The construct for the Kudzu isoprene synthase was used as control inthis example. The plasmids were transformed into the E. coli expressionhost BL21(DE3)plysS and transformants were grown in 0.6 ml TM3 medium.The recipe for TM3 medium is as follows: K₂HPO₄ (13.6 g/l) KH₂PO₄ (13.6g/l), MgSO₄*7H₂O (2 g/L) Citric Acid Monohydrate (2 g/L) Ferric AmmoniumCitrate (0.3 g/L) (NH₄)₂SO₄ (3.2 g/L) yeast extract (0.2 g/L) 1 ml of1000× Trace Elements solution, pH adjusted to 6.8 with ammoniumhydroxide qs to volume with sterile DI H₂O and filter sterilized with a0.22 micron filter. The recipe for 1000× Trace Elements solution is asfollows: Citric Acids*H₂O (40 g/L), MnSO₄*H₂O (30 g/L), NaCl (10 g/L),FeSO₄*7H₂O (1 g/L), CoCl₂*6H₂O (1 g/L), ZnSO₄*7H₂O (1 g/L), CuSO₄*5H₂O(100 mg/L), H₃BO₃ (100 mg/L), NaMoO₄*2H₂O (100 mg/L). Each component wasdissolved one at a time in DI H₂O, pH adjusted to 3.0 with HCl/NaOH, qsto volume and filter sterilized with a 0.22 micron filter.

The cultures were induced with 400 μM IPTG and growth was continued toOD₆₀₀ of about 5. Aliquots of culture were transferred to a deep wellglass plate and wells were sealed with aluminum plate sealer. The platewas incubated at 25° C. for 30 minutes with shaking at 450 rpm. Thereactions were heat inactivated by raising the temperature to 70° C. for5 minutes. Whole cell head space was measured by the GCMS method asdescribed in Example 1, Part II.

K_(m) values were obtained from cultures grown in similar manner butcells were harvested and lysed by a freeze/thaw lysozyme protocol. Avolume of 400 μl, of culture was transferred into a new 96-well plate(Perkin Elmer, Catalog No. 6008290) and cells were harvested bycentrifugation in a Beckman Coulter Allegra 6R centrifuge at 2500×g. Thepellet was resuspended in 200 mL of hypotonic buffer (5 mM MgCL₂, 5 mMTris HCl, 5 mM DTT pH 8.0) and the plate was frozen at −80° C. for aminimum time of 60 minutes. Cell lysate was prepared by thawing theplate and adding 32 mL of isoprene synthase DMAPP assay buffer (57 mMTris HCl, 19 mM MgCl₂, 74 mg/mL DNase I (Sigma Catalog No. DN-25),2.63×10⁵ U/mL of ReadyLyse lysozyme solution (Epicentre Catalog No.R1802M), and 5 mg/mL of molecular biology grade BSA. The plate wasincubated with shaking at 25° C. for 30 minutes and then placed on ice.DMAPP and lysate were added at desired concentration in a sealed deepwell glass block for the whole cell head space assay described above.The reactions were allowed to proceed for 1 hour and then terminated bythe heat step described above and head space activity was measured alsoas described.

In an alternate approach, the activity of the enzymes was measured fromcells cultured in 25 mL volume and induced similarly as described above.Cells were harvested by centrifugation and the pellets were lysed byFrench pressing in buffer consisting of 50% glycerol mixed 1:1 with 20mM Tris/HCl pH 7.4, 20 mM MgCl₂, 200 mM KCl, 1 mM DTT. A lysate volumeof 25 μL was assayed for isoprene synthase activity in 2 mL screw capvials containing 75 μL of assay buffer (66.6 mM Tris/HCl pH 8, 6.66 mMDMAPP, 43 mM, MgCl₂). The reaction was incubated for 15 minutes at 30°C. and was quenched by the addition of 100 μL of 250 mM EDTA through theseptum of the vial. Isoprene was measured by GC/MS as described inExample 1, Part II.

All methods for the determination of activity showed that the poplarenzyme derived from the pure bred poplars were several-fold higher thanthe Populus [alba×tremula]. FIGS. 138 and 139 showed these results forthe whole cell head space assay and the DMAPP assay, respectively, andsurprisingly indicate that enzymes from P. nigra, P. tremuloides, P.trichocarpa, and P. alba all had significantly higher activity thanhybrid [P. alba×P. tremula].

The DMAPP assay was performed as follows: a volume of 400 μL of culturewas transferred into a new 96-well plate (Perkin Elmer, Catalog No.6008290) and cells were harvested by centrifugation in a Beckman CoulterAllegra 6R centrifuge at 2500×g. The pellet was resuspended in 200 mL ofhypotonic buffer (5 mM MgCL₂, 5 mM Tris HCl, 5 mM DTT pH 8.0) and theplate was frozen at −80° C. for a minimum time of 60 minutes. Celllysate was prepared by thawing the plate and adding 32 mL of isoprenesynthase DMAPP assay buffer (57 mM Tris HCl, 19 mM MgCl₂, 74 mg/mL DNaseI (Sigma Catalog No. DN-25), 2.63×10⁵ U/mL of ReadyLyse lysozymesolution (Epicentre Catalog No. R1802M), and 5 mg/mL of molecularbiology grade BSA. The plate was incubated with shaking at 25° C. for 30minutes and then placed on ice. For isoprene production an 80 mL aliquotof lysate was transferred to a 96-deep well glass plate (Zinsser CatalogNo. 3600600) and 20 mL of a 10 mM DMAPP solution in 100 mM KHPO₄, pH 8.2(Cayman Chemical Catalog No. 63180) was added. The plate was sealed withan aluminum plate seal (Beckman Coultor Catalog No. 538619) andincubated with shaking at 30° C. for 60 minutes. The enzymatic reactionswere terminated by heating the glass block (70° C. for 5 minutes). Thecell head space of each well was quantitatively analyzed as described inExample 1, Part II.

Notably, P. alba, P. tremuloides, P. trichocarpa had higher activitythan the isoprene synthase from Kudzu. The enzyme from P. alba wasexpressed with the greatest activity of all enzymes tested. The higheractivities observed with the cell lysate compared to the whole cell headspace assay was likely due to limitations in DMAPP, the substrate forthese enzymes, delivered by the endogenous deoxyxylulose 5-phosphate(DXP) pathway of the cell.

K_(m) kinetic parameter was measured to be about 2 to 3 mM for allenzymes for which the value was determined.

Example 3 Production of Isoprene in Panteoa citrea ExpressingRecombinant Kudzu Isoprene Synthase

The pTrcKudzu and pCL-lac Kudzu plasmids described in Example 1 wereelectroporated into P. citrea (U.S. Pat. No. 7,241,587). Transformantswere selected on LA containing carbenicillin (200 μg/ml) orspectinomycin (50 μg/ml) respectively. Production of isoprene from shakeflasks and determination of the amount of isoprene produced wasperformed as described in Example 1 for E. coli strains expressingrecombinant kudzu isoprene synthase. Results are shown in FIG. 10.

Example 4 Production of Isoprene in Bacillus subtilis ExpressingRecombinant Kudzu Isoprene Synthase

I. Construction of a B. subtilis Replicating Plasmid for the Expressionof Kudzu Isoprene Synthase

The kudzu isoprene synthase gene was expressed in Bacillus subtilisaprEnprE Pxyl-comK strain (BG3594comK) using a replicating plasmid (0519with a chloramphenicol resistance cassette) under control of the aprEpromoter. The isoprene synthase gene, the aprE promoter and thetranscription terminator were amplified separately and fused using PCR.The construct was then cloned into pBSl9 and transformed into B.subtilis.

a) Amplification of the aprE Promoter

The aprE promoter was amplified from chromosomal DNA from Bacillussubtilis using the following primers:

CF 797 (+) Start aprE promoter MfeI (SEQ ID NO: 53)5′-GACATCAATTGCTCCATTTTCTTCTGCTATC CF 07-43 (−) Fuse aprE promoter toKudzu ispS (SEQ ID NO: 54) 5′-ATTGAGAAGAGGTCGCACACACTCTTTACCCTCTCCTTTTA

b) Amplification of the Isoprene Synthase Gene

The kudzu isoprene synthase gene was amplified from plasmid pTrcKudzu(SEQ ID NO:2). The gene had been codon optimized for E. coli andsynthesized by DNA 2.0. The following primers were used:

CF 07-42 (+) Fuse the aprE promoter to kudzu isoprene synthase gene (GTGstart codon) (SEQ ID NO: 55)5′-TAAAAGGAGAGGGTAAAGAGTGTGTGCGACCTCTTCTCAAT CF 07-45 (−) Fuse the3′ end of kudzu isoprene synthase gene to the terminator (SEQ ID NO: 56)5′-CCAAGGCCGGTTTTTTTTAGACATACATCAGCTGGTTAATC

c) Amplification of the Transcription Terminator

The terminator from the alkaline serine protease of Bacillusamyliquefaciens was amplified from a previously sequenced plasmidpJHPms382 using the following primers:

CF 07-44 (+) Fuse the 3′ end of kudzu isoprene synthase to theterminator (SEQ ID NO: 57) 5′-GATTAACCAGCTGATGTATGTCTAAAAAAAACCGGCCTTGGCF 07-46 (−) End of B. amyliquefaciens terminator (BamHI) (SEQ ID NO:58) 5′-GACATGACGGATCCGATTACGAATGCCGTCTC

The kudzu fragment was fused to the terminator fragment using PCR withthe following primers:

CF 07-42 (+) Fuse the aprE promoter to kudzuisoprene synthase gene (GTG start codon) (SEQ ID NO: 55)5′-TAAAAGGAGAGGGTAAAGAGTGTGTGCGACCTCTTCTCAATCF 07-46 (−) End of B. amyliquefaciens terminator (BamHI) 5′-GACATGACGGATCCGATTACGAATGCCGTCTC (SEQ ID NO: 58)

The kudzu-terminator fragment was fused to the promoter fragment usingPCR with the following primers:

CF 797 (+) Start aprE promoter MfeI 5′- GACATCAATTGCTCCATTTTCTTCTGCTATC(SEQ ID NO: 53) CF 07-46 (−) End of B. amyliquefaciens terminator(BamHI) 5′- GACATGACGGATCCGATTACGAATGCCGTCTC (SEQ ID NO: 58)

The fusion PCR fragment was purified using a Qiagen kit and digestedwith the restriction enzymes MfeI and BamHI. This digested DNA fragmentwas gel purified using a Qiagen kit and ligated to a vector known aspBS19, which had been digested with EcoRI and BamHI and gel purified.

The ligation mix was transformed into E. coli Top 10 cells and colonieswere selected on LA+50 carbenicillin plates. A total of six colonieswere chosen and grown overnight in LB+50 carbenicillin and then plasmidswere isolated using a Qiagen kit. The plasmids were digested with EcoRIand BamHI to check for inserts and three of the correct plasmids weresent in for sequencing with the following primers:

CF 149 (+) EcoRI start of aprE promoter 5′-GACATGAATTCCTCCATTTTCTTCTGC(SEQ ID NO: 59) CF 847 (+) Sequence in pXX 049 (end of aprE promoter)5′-AGGAGAGGGTAAAGAGTGAG (SEQ ID NO: 60) CF 07-45 (−) Fuse the 3′end of kudzu isoprene synthase to the terminator (SEQ ID NO: 56)5′-CCAAGGCCGGTTTTTTTTAGACATACATCAGCTGGTTAATCCF 07-48 (+) Sequencing primer for kudzu isoprene synthase5′-CTTTTCCATCACCCACCTGAAG (SEQ ID NO: 61)CF 07-49 (+) Sequencing in kudzu isoprene synthase5′-GGCGAAATGGTCCAACAACAAAATTATC (SEQ ID NO: 62)

The plasmid designated pBS Kudzu #2 (FIGS. 52 and 12; SEQ ID NO:5) wascorrect by sequencing and was transformed into BG 3594 comK, a Bacillussubtilis host strain. Selection was done on LA+5 chloramphenicol plates.A transformant was chosen and struck to single colonies on LA+5chloramphenicol, then grown in LB+5 chloramphenicol until it reached anOD₆₀₀ of 1.5. It was stored frozen in a vial at −80° C. in the presenceof glycerol. The resulting strain was designated CF 443.

II. Production of Isoprene in Shake Flasks Containing B. subtilis CellsExpressing Recombinant Isoprene Synthase

Overnight cultures were inoculated with a single colony of CF 443 from aLA+Chloramphenicol (Cm, 25 μg/ml). Cultures were grown in LB+Cm at 37°C. with shaking at 200 rpm. These overnight cultures (1 ml) were used toinoculate 250 ml baffled shake flasks containing 25 ml Grants II mediaand chloramphenicol at a final concentration of 25 μg/ml. Grants IIMedia recipe was 10 g soytone, 3 ml 1M K₂HPO₄, 75 g glucose, 3.6 g urea,100 ml 10×MOPS, q.s. to 1 L with H₂O, pH 7.2; 10×MOPS recipe was 83.72 gMOPS, 7.17 g tricine, 12 g KOH pellets, 10 ml 0.276M K₂SO₄ solution, 10ml 0.528M MgCl₂ solution, 29.22 g NaCl, 100 ml 100× micronutrients, q.s.to 1 L with H₂O; and 100× micronutrients recipe was 1.47 g CaCl₂*2H₂O,0.4 g FeSO₄.7H₂O, 0.1 g MnSO₄*H₂O, 0.1 g ZnSO₄*H₂O, 0.05 g CuCl₂*2H₂O,0.1 g CoCl₂*6H₂O, 0.1 g Na₂MoO₄.2H₂O, q.s. to 1 L with H₂O, Shake flaskswere incubated at 37° C. and samples were taken at 18, 24, and 44 hours.At 18 hours the headspaces of CF₄₄₃ and the control strain were sampled.This represented 18 hours of accumulation of isoprene. The amount ofisoprene was determined by gas chromatography as described in Example 1.Production of isoprene was enhanced significantly by expressingrecombinant isoprene synthase (FIG. 11).

III. Production of Isoprene by CF443 in 14 L Fermentation

Large scale production of isoprene from B. subtilis containing therecombinant kudzu isoprene synthase gene on a replication plasmid wasdetermined from a fed-batch culture. Bacillus strain CF 443, expressinga kudzu isoprene synthase gene, or control stain which does not expressa kudzu isoprene synthase gene were cultivated by conventional fed-batchfermentation in a nutrient medium containing soy meal (Cargill), sodiumand potassium phosphate, magnesium sulfate and a solution of citricacid, ferric chloride and manganese chloride. Prior to fermentation themedia is macerated for 90 minutes using a mixture of enzymes includingcellulases, hemicellulases and pectinases (see, WO95/04134). 14-L batchfermentations are fed with 60% wt/wt glucose (Cargill DE99 dextrose, ADMVersadex greens or Danisco invert sugar) and 99% wt/wt oil (WesternFamily soy oil, where the 99% wt/wt is the concentration of oil beforeit was added to the cell culture medium). Feed was started when glucosein the batch was non-detectable. The feed rate was ramped over severalhours and was adjusted to add oil on an equal carbon basis. The pH wascontrolled at 6.8-7.4 using 28% w/v ammonium hydroxide. In case offoaming, antifoam agent was added to the media. The fermentationtemperature was controlled at 37° C. and the fermentation culture wasagitated at 750 rpm. Various other parameters such as pH, D0%, airflow,and pressure were monitored throughout the entire process. The DO % ismaintained above 20. Samples were taken over the time course of 36 hoursand analyzed for cell growth (OD₅₅₀) and isoprene production. Results ofthese experiments are presented in FIGS. 53A and 53B.

IV. Integration of the Kudzu Isoprene Synthase (ispS) in B. subtilis.

The kudzu isoprene synthase gene was cloned in an integrating plasmid(pJH101-cmpR) under the control of the aprE promoter. Under theconditions tested, no isoprene was detected.

Example 5 Production of Isoprene in Trichoderma

I. Construction of Vectors for Expression of the Kudzu Isoprene Synthasein Trichoderma reesei

The Yarrowia lipolytica codon-optimized kudzu IS gene was synthesized byDNA 2.0 (SEQ ID NO:6) (FIG. 13). This plasmid served as the template forthe following PCR amplification reaction: 1 μl plasmid template (20ng/ul), 1 μl Primer EL-945 (10 μM)5′-GCTTATGGATCCTCTAGACTATTACACGTACATCAATTGG (SEQ ID NO:63), 1 μl PrimerEL-965 (10 μM) 5′-CACCATGTGTGCAACCTCCTCCCAGTTTAC (SEQ ID NO:64), 1 μldNTP (10 mM), 5 μl 10×PfuUltra II Fusion HS DNA Polymerase Buffer, 1 μlPfuUltra II Fusion HS DNA Polymerase, 40 μl water in a total reactionvolume of 50 μl. The forward primer contained an additional 4nucleotides at the 5′-end that did not correspond to the Y. lipolyticacodon-optimized kudzu isoprene synthase gene, but was required forcloning into the pENTR/D-TOPO vector. The reverse primer contained anadditional 21 nucleotides at the 5′-end that did not correspond to theY. lipolytica codon-optimized kudzu isoprene synthase gene, but wereinserted for cloning into other vector backbones. Using the MJ ResearchPTC-200 Thermocycler, the PCR reaction was performed as follows: 95° C.for 2 minutes (first cycle only), 95° C. for 30 seconds, 55° C. for 30seconds, 72° C. for 30 seconds (repeat for 27 cycles), 72° C. for 1minute after the last cycle. The PCR product was analyzed on a 1.2%E-gel to confirm successful amplification of the Y. lipolyticacodon-optimized kudzu isoprene synthase gene.

The PCR product was then cloned using the TOPO pENTR/D-TOPO Cloning Kitfollowing manufacturer's protocol: 1 μl PCR reaction, 1 μl Saltsolution, 1 μl TOPO pENTR/D-TOPO vector and 3 μl water in a totalreaction volume of 6 μl. The reaction was incubated at room temperaturefor 5 minutes. One microliter of TOPO reaction was transformed intoTOP10 chemically competent E. coli cells. The transformants wereselected on LA+50 μg/mlkanamycin plates. Several colonies were pickedand each was inoculated into a 5 ml tube containing LB+50 μg/mlkanamycinand the cultures grown overnight at 37° C. with shaking at 200 rpm.Plasmids were isolated from the overnight culture tubes using QIAprepSpin Miniprep Kit, following manufacturer's protocol. Several plasmidswere sequenced to verify that the DNA sequence was correct.

A single pENTR/D-TOPO plasmid, encoding a Y. lipolytica codon-optimizedkudzu isoprene synthase gene, was used for Gateway Cloning into acustom-made pTrex3g vector. Construction of pTrex3g is described in WO2005/001036 A2. The reaction was performed following manufacturer'sprotocol for the Gateway LR Clonase II Enzyme Mix Kit (Invitrogen): 1 μlY. lipolytica codon-optimized kudzu isoprene synthase gene pENTR/D-TOPOdonor vector, 1 μl pTrex3g destination vector, 6 μl TE buffer, pH 8.0 ina total reaction volume of 8 μl. The reaction was incubated at roomtemperature for 1 hour and then 1 μl proteinase K solution was added andthe incubation continued at 37° C. for 10 minutes. Then 1 μl of reactionwas transformed into TOP10 chemically competent E. coli cells. Thetransformants were selected on LA+50 μg/ml carbenicillin plates. Severalcolonies were picked and each was inoculated into a 5 ml tube containingLB+50 μg/ml carbenicillin and the cultures were grown overnight at 37°C. with shaking at 200 rpm. Plasmids were isolated from the overnightculture tubes using QIAprep Spin Miniprep Kit (Qiagen, Inc.), followingmanufacturer's protocol. Several plasmids were sequenced to verify thatthe DNA sequence was correct.

Biolistic transformation of Y. lipolytica codon-optimized kudzu isoprenesynthase pTrex3g plasmid (FIG. 14) into a quad delete Trichoderma reeseistrain was performed using the Biolistic PDS-1000/HE Particle DeliverySystem (see WO 2005/001036 A2). Isolation of stable transformants andshake flask evaluation was performed using protocol listed in Example 11of patent publication WO 2005/001036 A2.

II. Production of Isoprene in Recombinant Strains of T. reesei

One ml of 15 and 36 hour old cultures of isoprene synthase transformantsdescribed above were transferred to head space vials. The vials weresealed and incubated for 5 hours at 30° C. Head space gas was measuredand isoprene was identified by the method described in Example 1. Two ofthe transformants showed traces of isoprene. The amount of isoprenecould be increased by a 14 hour incubation. The two positive samplesshowed isoprene at levels of about 0.5 μg/L for the 14 hour incubation.The untransformed control showed no detectable levels of isoprene. Thisexperiment shows that T. reesei is capable of producing isoprene fromendogenous precursor when supplied with an exogenous isoprene synthase.

Example 6 Production of Isoprene in Yarrowia

I. Construction of Vectors for Expression of the Kudzu Isoprene Synthasein Yarrowia lipolytica.

The starting point for the construction of vectors for the expression ofthe kudzu isoprene synthase gene in Yarrowia lipolytica was the vectorpSPZ1(MAP29Spb). The complete sequence of this vector (SEQ ID NO:7) isshown in FIG. 15.

The following fragments were amplified by PCR using chromosomal DNA of aY. lipolytica strain GICC 120285 as the template: a promotorless form ofthe URA3 gene, a fragment of 18S ribosomal RNA gene, a transcriptionterminator of the Y. lipolytica XPR2 gene and two DNA fragmentscontaining the promoters of XPR2 and ICL1 genes. The following PCRprimers were used:

ICL1 3 (SEQ ID NO: 65)5′-GGTGAATTCAGTCTACTGGGGATTCCCAAATCTATATATACTGCAGG TGAC ICL1 55′-GCAGGTGGGAAACTATGCACTCC (SEQ ID NO: 66) XPR 3 5′-CCTGAATTCTGTTGGATTGGAGGATTGGATAGTGGG (SEQ ID NO: 67) XPR 55′-GGTGTCGACGTACGGTCGAGCTTATTGACC (SEQ ID NO: 68) XPRT35′-GGTGGGCCCGCATTTTGCCACCTACAAGCCAG (SEQ ID NO: 69) XPRT 5(SEQ ID NO: 70) 5′-GGTGAATTCTAGAGGATCCCAACGCTGTTGCCTACAACGG Y18S3 5′-GGTGCGGCCGCTGTCTGGACCTGGTGAGTTTCCCCG (SEQ ID NO: 71) Y18S 5 5′-GGTGGGCCCATTAAATCAGTTATCGTTTATTTGATAG (SEQ ID NO: 72) YURA3 5′-GGTGACCAGCAAGTCCATGGGTGGTTTGATCATGG (SEQ ID NO: 73) YURA 50 5′-GGTGCGGCCGCCTTTGGAGTACGACTCCAACTATG (SEQ ID NO: 74) YURA 515′-GCGGCCGCAGACTAAATTTATTTCAGTCTCC (SEQ ID NO: 75)

For PCR amplification the PfuUltraII polymerase (Stratagene),supplier-provided buffer and dNTPs, 2.5 μM primers and the indicatedtemplate DNA were used as per the manufacturer's instructions. Theamplification was done using the following cycle: 95° C. for 1 min;34×(95° C. for 30 sec; 55° C. for 30 sec; 72° C. for 3 min) and 10 minat 72° C. followed by a 4° C. incubation.

Synthetic DNA molecules encoding the kudzu isoprene synthase gene,codon-optimized for expression in Yarrowia, was obtained from DNA 2.0(FIG. 16; SEQ ID NO:8). Full detail of the construction scheme of theplasmids pYLA(KZ1) and pYLI(KZ1) carrying the synthetic kudzu isoprenesynthase gene under control of XPR2 and ICL1 promoters respectively ispresented in FIG. 18. Control plasmids in which a mating factor gene(MAP29) is inserted in place of an isoprene synthase gene were alsoconstructed (FIGS. 18E and 18F).

A similar cloning procedure can be used to express a poplar (Populusalba×Populus tremula) isoprene synthase gene. The sequence of the poplarisoprene is described in Miller B. et al. (2001) Planta 213, 483-487 andshown in FIG. 17 (SEQ ID NO:9). A construction scheme for the generationthe plasmids pYLA(POP1) and pYLI(POP1) carrying synthetic poplarisoprene synthase gene under control of XPR2 and ICL1 promotersrespectively is presented in FIGS. 18A and B.

II. Production of Isoprene by Recombinant Strains of Y. lipolytica.

Vectors pYLA(KZ1), pYLI(KZ1), pYLA(MAP29) and pYLI(MAP29) were digestedwith SacII and used to transform the strain Y. lipolytica CLIB 122 by astandard lithium acetate/polyethylene glycol procedure to uridineprototrophy. Briefly, the yeast cells grown in YEPD (1% yeast extract,2% peptone, 2% glucose) overnight, were collected by centrifugation(4000 rpm, 10 min), washed once with sterile water and suspended in 0.1M lithium acetate, pH 6.0. Two hundred μl aliquots of the cellsuspension were mixed with linearized plasmid DNA solution (10-20 μg),incubated for 10 minutes at room temperature and mixed with 1 ml of 50%PEG 4000 in the same buffer. The suspensions were further incubated for1 hour at room temperature followed by a 2 minutes heat shock at 42° C.Cells were then plated on SC his leu plates (0.67% yeast nitrogen base,2% glucose, 100 mg/L each of leucine and histidine). Transformantsappeared after 3-4 days of incubation at 30° C.

Three isolates from the pYLA(KZ1) transformation, three isolates fromthe pYLI(KZ1) transformation, two isolates from the pYLA(MAP29)transformation and two isolates from the pYLI(MAP29) transformation weregrown for 24 hours in YEP7 medium (1% yeast extract, 2% peptone, pH 7.0)at 30° C. with shaking Cells from 10 ml of culture were collected bycentrifugation, resuspended in 3 ml of fresh YEP7 and placed into 15 mlscrew cap vials. The vials were incubated overnight at room temperaturewith gentle (60 rpm) shaking. Isoprene content in the headspace of thesevials was analyzed by gas chromatography using mass-spectrometricdetector as described in Example 1. All transformants obtained withpYLA(KZ1) and pYLI(KZ1) produced readily detectable amounts of isoprene(0.5 μg/L to 1 μg/L, FIG. 20). No isoprene was detected in the headspaceof the control strains carrying phytase gene instead of an isoprenesynthase gene.

Example 7 Production of Isoprene in E. coli Expressing Kudzu IsopreneSynthase and idi, or dxs, or idi and dxs

I. Construction of Vectors Encoding Kudzu Isoprene Synthase and idi, ordxs, or idi and dxs for the Production of Isoprene in E. colii) Construction of pTrcKudzuKan

The bla gene of pTrcKudzu (described in Example 1) was replaced with thegene conferring kanamycin resistance. To remove the bla gene, pTrcKudzuwas digested with BspHI, treated with Shrimp Alkaline Phosphatase (SAP),heat killed at 65° C., then end-filled with Klenow fragment and dNTPs.The 5 kbp large fragment was purified from an agarose gel and ligated tothe kan^(r) gene which had been PCR amplified from pCR-Blunt-II-TOPOusing primers MCM22 5′-GATCAAGCTTAACCGGAATTGCCAGCTG (SEQ ID NO:76) andMCM23 5′-GATCCGATCGTCAGAAGAACTCGTCAAGAAGGC (SEQ ID NO:77), digested withHindIII and Pvul, and end-filled. A transformant carrying a plasmidconferring kanamycin resistance (pTrcKudzuKan) was selected on LAcontaining kanamycin 50 μg/ml.

ii) Construction of pTrcKudzu yIDI Kan

pTrcKudzuKan was digested with PstI, treated with SAP, heat killed andgel purified. It was ligated to a PCR product encoding idi from S.cerevisiae with a synthetic RBS. The primers for PCR were NsiI-YIDI 1 F5′-CATCAATGCATCGCCCTTAGGAGGTAAAAAAAAATGAC (SEQ ID NO:78) and PstI-YIDI 1R 5′-CCTTCTGCAGGACGCGTTGTTATAGC (SEQ ID NO:79); and the template was S.cerevisiae genomic DNA. The PCR product was digested with NsiI and PstIand gel purified prior to ligation. The ligation mixture was transformedinto chemically competent TOP10 cells and selected on LA containing 50μg/mlkanamycin. Several transformants were isolated and sequenced andthe resulting plasmid was called pTrcKudzu-yIDI(kan) (FIGS. 34 and 35;SEQ ID NO:16).

iii) Construction of pTrcKudzu DXS Kan

Plasmid pTrcKudzuKan was digested with PstI, treated with SAP, heatkilled and gel purified. It was ligated to a PCR product encoding dxsfrom E. coli with a synthetic RBS. The primers for PCR were MCM135′-GATCATGCATTCGCCCTTAGGAGGTAAAAAAACATGAGTTTTGATATTGCCAAATACCC G (SEQ IDNO:80) and MCM14 5′-CATGCTGCAGTTATGCCAGCCAGGCCTTGAT (SEQ ID NO:81); andthe template was E. coli genomic DNA. The PCR product was digested withNsiI and PstI and gel purified prior to ligation. The resultingtransformation reaction was transformed into TOP10 cells and selected onLA with kanamycin 50 μg/ml. Several transformants were isolated andsequenced and the resulting plasmid was called pTrcKudzu-DXS(kan) (FIGS.36 and 37; SEQ ID NO:17).

iv) Construction of pTrcKudzu-yIDI-dxs (kan)

pTrcKudzu-yIDI(kan) was digested with PstI, treated with SAP, heatkilled and gel purified. It was ligated to a PCR product encoding E.coli dxs with a synthetic RBS (primers MCM135′-GATCATGCATTCGCCCTTAGGAGGTAAAAAAACATGAGTTTTGATATTGCCAAATACCC G (SEQ IDNO:80) and MCM14 5′-CATGCTGCAGTTATGCCAGCCAGGCCTTGAT (SEQ ID NO:81);template TOP10 cells) which had been digested with NsiI and PstI and gelpurified. The final plasmid was called pTrcKudzu-yIDI-dxs (kan) (FIGS.21 and 22; SEQ ID NO:10).

v) Construction of pCL PtrcKudzu

A fragment of DNA containing the promoter, structural gene andterminator from Example 1 above was digested from pTrcKudzu using Sspland gel purified. It was ligated to pCL1920 which had been digested withPvuII, treated with SAP and heat killed. The resulting ligation mixturewas transformed into TOP10 cells and selected in LA containingspectinomycin 50 μg/ml. Several clones were isolated and sequenced andtwo were selected. pCL PtrcKudzu and pCL PtrcKudzu (A3) have the insertin opposite orientations (FIGS. 38-41; SEQ ID NOs:18-19).

vi) Construction of pCL PtrcKudzu yIDI

The NsiI-PstI digested, gel purified, IDI PCR amplicon from (ii) abovewas ligated into pCL PtrcKudzu which had been digested with PstI,treated with SAP, and heat killed. The ligation mixture was transformedinto TOP10 cells and selected in LA containing spectinomycin 50 μg/ml.Several clones were isolated and sequenced and the resulting plasmid iscalled pCL PtrcKudzu yIDI (FIGS. 42 and 43; SEQ ID NO:20).

vii) Construction of pCL PtrcKudzu DXS

The NsiI-PstI digested, gel purified, DXS PCR amplicon from (iii) abovewas ligated into pCL PtrcKudzu (A3) which had been digested with PstI,treated with SAP, and heat killed. The ligation mixture was transformedinto TOP10 cells and selected in LA containing spectinomycin 50 μg/ml.Several clones were isolated and sequenced and the resulting plasmid iscalled pCL PtrcKudzu DXS (FIGS. 44 and 45; SEQ ID NO:21).

II. Measurement of Isoprene in Headspace from Cultures Expressing KudzuIsoprene Synthase, idi, and/or dxs at Different Copy Numbers.

Cultures of E. coli BL21(λDE3) previously transformed with plasmidspTrcKudzu(kan) (A), pTrcKudzu-yIDI kan (B), pTrcKudzu-DXS kan (C),pTrcKudzu-yIDI-DXS kan (D) were grown in LB kanamycin 50 μg/mL. Culturesof pCL PtrcKudzu (E), pCL PtrcKudzu, pCL PtrcKudzu-yIDI (F) and pCLPtrcKudzu-DXS (G) were grown in LB spectinomycin 50 μg/mL. Cultures wereinduced with 400 μM IPTG at time 0 (OD₆₀₀ approximately 0.5) and samplestaken for isoprene headspace measurement (see Example 1). Results areshown in FIG. 23A-23G.

Plasmid pTrcKudzu-yIDI-dxs (kan) was introduced into E. coli strain BL21by transformation. The resulting strain BL21/pTrc Kudzu IDI DXS wasgrown overnight in LB containing kanamycin (50 μg/ml) at 20° C. and usedto inoculate shake flasks of TM3 (13.6 g K₂PO₄, 13.6 g KH₂PO₄, 2.0 gMgSO₄.7H₂O), 2.0 g citric acid monohydrate, 0.3 g ferric ammoniumcitrate, 3.2 g (NH₄)₂SO₄, 0.2 g yeast extract, 1.0 ml 1000× ModifiedTrace Metal Solution, adjusted to pH 6.8 and q.s. to H₂O, and filtersterilized) containing 1% glucose. Flasks were incubated at 30° C. untilan OD₆₀₀ of 0.8 was reached, and then induced with 400 μM IPTG. Sampleswere taken at various times after induction and the amount of isoprenein the head space was measured as described in Example 1. Results areshown in FIG. 23H.

III. Production of Isoprene from Biomass in E. coli/pTrcKudzu yIDI DXS

The strain BL21 pTrcKudzuIDIDXS was tested for the ability to generateisoprene from three types of biomass; bagasse, corn stover and soft woodpulp with glucose as a control. Hydrolysates of the biomass wereprepared by enzymatic hydrolysis (Brown, L. and Torget, R., 1996, NRELstandard assay method Lap-009 “Enzymatic Saccharification ofLignocellulosic Biomass”) and used at a dilution based upon glucoseequivalents. In this example, glucose equivalents were equal to 1%glucose. A single colony from a plate freshly transformed cells of BL21(DE3) pTrcKudzu yIDI DXS (kan) was used to inoculate 5 ml of LB pluskanamycin (50 μg/ml). The culture was incubated overnight at 25° C. withshaking. The following day the overnight culture was diluted to an OD₆₀₀of 0.05 in 25 ml of TM3+0.2% YE+1% feedstock. The feedstock was cornstover, bagasse, or softwood pulp. Glucose was used as a positivecontrol and no glucose was used as a negative control. Cultures wereincubated at 30° C. with shaking at 180 rpm. The culture was monitoredfor OD₆₀₀ and when it reached an OD₆₀₀ of ˜0.8, cultures were analyzedat 1 and 3 hours for isoprene production as described in Example 1.Cultures are not induced. All cultures containing added feedstockproduce isoprene equivalent to those of the glucose positive control.Experiments were done in duplicate and are shown in FIG. 46.

IV. Production of Isoprene from Invert Sugar in E. coli/pTrcKudzuIDIDXS

A single colony from a plate freshly transformed cells of BL21(ADE3)/pTrcKudzu yIDI DXS (kan) was used to inoculate 5 mL ofLB+kanamycin (50 μg/ml). The culture was incubated overnight at 25° C.with shaking. The following day the overnight culture was diluted to anOD₆₀₀ of 0.05 in 25 ml of TM3+0.2% YE+1% feedstock. Feedstock wasglucose, inverted glucose or corn stover. The invert sugar feedstock(Danisco Invert Sugar) was prepared by enzymatically treating sucrosesyrup. AFEX corn stover was prepared as described below (Part V). Thecells were grown at 30° C. and the first sample was measured when thecultures reached an OD₆₀₀˜0.8-1.0 (0 hour). The cultures were analyzedfor growth as measured by OD₆₀₀ and for isoprene production as inExample 1 at 0, 1 and 3 hours. Results are shown in FIG. 47.

V. Preparation of Hydrolysate from AFEX Pretreated Corn Stover

AFEX pretreated corn stover was obtained from Michigan BiotechnologyInstitute. The pretreatment conditions were 60% moisture, 1:1 ammonialoading, and 90° C. for 30 minutes, then air dried. The moisture contentin the AFEX pretreated corn stover was 21.27%. The contents of glucanand xylan in the AFEX pretreated corn stover were 31.7% and 19.1% (drybasis), respectively. The saccharification process was as follows; 20 gof AFEX pretreated corn stover was added into a 500 ml flask with 5 mlof 1 M sodium citrate buffer pH 4.8, 2.25 ml of Accellerase 1000, 0.1 mlof Grindamyl H121 (Danisco xylanase product from Aspergillus niger forbread-making industry), and 72.65 ml of DI water. The flask was put inan orbital shaker and incubated at 50° C. for 96 hours. One sample wastaken from the shaker and analyzed using HPLC. The hydrolysate contained38.5 g/l of glucose, 21.8 g/l of xylose, and 10.3 g/l of oligomers ofglucose and/or xylose.

VI. The Effect of Yeast Extract on Isoprene Production in E. coli Grownin Fed-Batch Culture

Fermentation was performed at the 14-L scale as previously describedwith E. coli cells containing the pTrcKudzu yIDI DXS plasmid describedabove. Yeast extract (Bio Springer, Montreal, Quebec, Canada) was fed atan exponential rate. The total amount of yeast extract delivered to thefermentor was varied between 70-830 g during the 40 hour fermentation.Optical density of the fermentation broth was measured at a wavelengthof 550 nm. The final optical density within the fermentors wasproportional to the amount of yeast extract added (FIG. 48A). Theisoprene level in the off-gas from the fermentor was determined aspreviously described. The isoprene titer increased over the course ofthe fermentation (FIG. 48B). The amount of isoprene produced waslinearly proportional to the amount of fed yeast extract (FIG. 48C).

VII. Production of Isoprene in 500 L Fermentation of pTrcKudzu DXS yIDI

A 500 liter fermentation of E. coli cells with a kudzu isoprenesynthase, S. cerevisiae IDI, and E. coli DXS nucleic acids (E. coli BL21(λDE3) pTrc Kudzu dxs yidi) was used to produce isoprene. The levels ofisoprene varied from 50 to 300 μg/L over a time period of 15 hours. Onthe basis of the average isoprene concentrations, the average flowthrough the device and the extent of isoprene breakthrough, the amountof isoprene collected was calculated to be approximately 17 g.

VIII. Production of Isoprene in 500 L Fermentation of E. coli Grown inFed-Batch Culture

Medium Recipe (Per Liter Fermentation Medium):

K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2 g, citric acid monohydrate 2 g, ferricammonium citrate 0.3 g, yeast extract 0.5 g, 1000× Modified Trace MetalSolution 1 ml. All of the components were added together and dissolvedin diH₂O. This solution was autoclaved. The pH was adjusted to 7.0 withammonium gas (NH₃) and q.s. to volume. Glucose 10 g, thiamine*HCl 0.1 g,and antibiotic were added after sterilization and pH adjustment.

1000× Modified Trace Metal Solution:

Citric Acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g, FeSO₄*7H₂O 1 g,CoCl₂*6H₂O 1 g, ZnSO*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃ 100 mg,NaMoO₄*2H₂O 100 mg. Each component is dissolved one at a time in DI H₂O,pH to 3.0 with HCl/NaOH, then q.s. to volume and filter sterilized with0.22 micron filter.

Fermentation was performed in a 500-L bioreactor with E. coli cellscontaining the pTrcKudzu yIDI DXS plasmid. This experiment was carriedout to monitor isoprene formation from glucose and yeast extract at thedesired fermentation pH 7.0 and temperature 30° C. An inoculum of E.coli strain taken from a frozen vial was prepared in soytone-yeastextract-glucose medium. After the inoculum grew to OD 0.15, measured at550 nm, 20 ml was used to inoculate a bioreactor containing 2.5-Lsoytone-yeast extract-glucose medium. The 2.5-L bioreactor was grown at30° C. to OD 1.0 and 2.0-L was transferred to the 500-L bioreactor.

Yeast extract (Bio Springer, Montreal, Quebec, Canada) and glucose werefed at exponential rates. The total amount of glucose and yeast extractdelivered to the bioreactor during the 50 hour fermentation was 181.2 kgand 17.6 kg, respectively. The optical density within the bioreactorover time is shown in FIG. 49A. The isoprene level in the off-gas fromthe bioreactor was determined as previously described. The isoprenetiter increased over the course of the fermentation (FIG. 49B). Thetotal amount of isoprene produced during the 50 hour fermentation was55.1 g and the time course of production is shown in FIG. 49C.

Example 8 Production of Isoprene in E. coli Expressing Kudzu IsopreneSynthase and Recombinant Mevalonic Acid Pathway Genes I. Cloning theLower MVA Pathway

The strategy for cloning the lower mevalonic pathway was as follows.Four genes of the mevalonic acid biosynthesis pathway; mevalonate kinase(MVK), phosphomevalonate kinase (PMK), diphosphomevalonte decarboxylase(MVD) and isopentenyl diphosphate isomerase genes were amplified by PCRfrom S. cerevisiae chromosomal DNA and cloned individually into the pCRBluntII TOPO plasmid (Invitrogen). In some cases, the idi gene wasamplified from E. coli chromosomal DNA. The primers were designed suchthat an E. coli consensus RBS (AGGAGGT (SEQ ID NO:82) or AAGGAGG (SEQ IDNO:83)) was inserted at the 5′ end, 8 by upstream of the start codon anda PstI site was added at the 3′ end. The genes were then cloned one byone into the pTrcHis2B vector until the entire pathway was assembled.

Chromosomal DNA from S. cerevisiae S288C was obtained from ATCC (ATCC204508D). The MVK gene was amplified from the chromosome of S.cerevisiae using primers MVKF(5′-AGGAGGTAAAAAAACATGTCATTACCGTTCTTAACTTCTGC, SEQ ID NO:84) andMVK-PstI-R (5′-ATGGCTGCAGGCCTATCGCAAATTAGCTTATGAAGTCCATGGTAAATTCGTG, SEQID NO:85) using PfuTurbo as per manufacturer's instructions. The correctsized PCR product (1370 bp) was identified by electrophoresis through a1.2% E-gel (Invitrogen) and cloned into pZeroBLUNT TOPO. The resultingplasmid was designated pMVK1. The plasmid pMVK1 was digested with Sadand Taql restriction endonucleases and the fragment was gel purified andligated into pTrcHis2B digested with Sad and BstBI. The resultingplasmid was named pTrcMVK1.

The second gene in the mevalonic acid biosynthesis pathway, PMK, wasamplified by PCR using primers: PstI-PMK1 R (5′-GAATTCGCCCTTCTGCAGCTACC,SEQ ID NO:86) and BsiHKA I-PMK1 F(5′-CGACTGGTGCACCCTTAAGGAGGAAAAAAACATGTCAG, SEQ ID NO:87). The PCRreaction was performed using Pfu Turbo polymerase (Stratagene) as permanufacturer's instructions. The correct sized product (1387 bp) wasdigested with PstI and BsiHKI and ligated into pTrcMVK1 digested withPstI. The resulting plasmid was named pTrcKK. The MVD and the idi geneswere cloned in the same manner. PCR was carried out using the primerpairs PstI-MVD 1 R (5′-GTGCTGGAATTCGCCCTTCTGCAGC, SEQ ID NO:88) andNsiI-MVD 1 F (5′-GTAGATGCATGCAGAATTCGCCCTTAAGGAGG, SEQ ID NO:89) toamplify the MVD gene and PstI-YIDI 1 R (5′-CCTTCTGCAGGACGCGTTGTTATAGC,SEQ ID NO:79) and NsiI-YIDI 1 F(5′-CATCAATGCATCGCCCTTAGGAGGTAAAAAAAAATGAC, SEQ ID NO:78) to amplify theyIDI gene. In some cases the IPP isomerase gene, idi from E. coli wasused. To amplify idi from E. coli chromosomal DNA, the following primerset was used: PstI-CIDI 1 R (5′-GTGTGATGGATATCTGCAGAATTCG, SEQ ID NO:90)and NsiI-CIDI 1 F (5′-CATCAATGCATCGCCCTTAGGAGGTAAAAAAACATG, SEQ IDNO:91). Template DNA was chromosomal DNA isolated by standard methodsfrom E. coli FM5 (WO 96/35796 and WO 2004/033646, which are each herebyincorporated by reference in their entireties, particularly with respectto isolation of nucleic acids). The final plasmids were named pKKDIy forthe construct encoding the yeast idi gene or pKKDIc for the constructencoding the E. coli idi gene. The plasmids were transformed into E.coli hosts BL21 for subsequent analysis. In some cases the isoprenesynthase from kudzu was cloned into pKKDIy yielding plasmid pKKDIyIS.

The lower MVA pathway was also cloned into pTrc containing a kanamycinantibiotic resistance marker. The plasmid pTrcKKDIy was digested withrestriction endonucleases ApaI and PstI, the 5930 by fragment wasseparated on a 1.2% agarose E-gel and purified using the Qiagen GelPurification kit according to the manufacturer's instructions. Theplasmid pTrcKudzuKan, described in Example 7, was digested withrestriction endonucleases ApaI and PstI, and the 3338 by fragmentcontaining the vector was purified from a 1.2% E-gel using the QiagenGel Purification kit. The 3338 by vector fragment and the 5930 by lowerMVA pathway fragment were ligated using the Roche Quick Ligation kit.The ligation mix was transformed into E. coli TOP10 cells andtranformants were grown at 37° C. overnight with selection on LAcontaining kanamycin (50 μg/ml). The transformants were verified byrestriction enzyme digestion and one was frozen as a stock. The plasmidwas designated pTrcKanKKDIy.

II. Cloning a Kudzu Isoprene Synthase Gene into pTrcKanKKDIy

The kudzu isoprene synthase gene was amplified by PCR from pTrcKudzu,described in Example 1, using primers MCM505′-GATCATGCATTCGCCCTTAGGAGGTAAAAAAACATGTGTGCGACCTCTTCTCAATTTAC T (SEQ IDNO:52) and MCM53 5′-CGGTCGACGGATCCCTGCAGTTAGACATACATCAGCTG (SEQ IDNO:50). The resulting PCR fragment was cloned into pCR2.1 andtransformed into E. coli TOP10. This fragment contains the codingsequence for kudzu isoprene synthase and an upstream region containing aRBS from E. coli. Transformants were incubated overnight at 37° C. withselection on LA containing carbenicillin (50 μg/ml). The correctinsertion of the fragment was verified by sequencing and this strain wasdesignated MCM93.

The plasmid from strain MCM93 was digested with restrictionendonucleases NsiI and PstI to liberate a 1724 by insert containing theRBS and kudzu isoprene synthase. The 1724 by fragment was separated on a1.2% agarose E-gel and purified using the Qiagen Gel Purification kitaccording to the manufacturer's instructions. Plasmid pTrcKanKKDIy wasdigested with the restriction endonuclease PstI, treated with SAP for 30minutes at 37° C. and purified using the Qiagen PCR cleanup kit. Theplasmid and kudzu isoprene synthase encoding DNA fragment were ligatedusing the Roche Quick Ligation kit. The ligation mix was transformedinto E. coli TOP10 cells and transformants were grown overnight at 37°C. with selection on LA containing Kanamycin at 50 μg/ml. The correcttransformant was verified by restriction digestion and the plasmid wasdesignated pTrcKKDyIkISKan (FIGS. 24 and 25; SEQ ID NO:11). This plasmidwas transformed into BL21(λDE3) cells (Invitrogen).

III. Isoprene Production from Mevalonate in E. coli Expressing theRecombinant Lower Mevalonate Pathway and Isoprene Synthase from Kudzu.

Strain BL21/pTrcKKDyIkISKan was cultured in MOPS medium (Neidhardt etal., (1974) J. Bacteriology 119:736-747) adjusted to pH 7.1 andsupplemented with 0.5% glucose and 0.5% mevalonic acid. A controlculture was also set up using identical conditions but without theaddition of 0.5% mevalonic acid. The culture was started from anovernight seed culture with a 1% inoculum and induced with 500 μM IPTGwhen the culture had reached an OD₆₀₀ of 0.3 to 0.5. The cultures weregrown at 30° C. with shaking at 250 rpm. The production of isoprene wasanalyzed 3 hours after induction by using the head space assay describedin Example 1. Maximum production of isoprene was 6.67×10⁻⁴mol/L_(broth)/OD₆₀₀/hr where L_(broth) is the volume of broth andincludes both the volume of the cell medium and the volume of the cells.The control culture not supplemented with mevalonic acid did not producemeasurable isoprene.

IV. Cloning the Upper MVA Pathway

The upper mevalonate biosynthetic pathway, comprising two genes encodingthree enzymatic activities, was cloned from Enterococcus faecalis. ThemvaE gene encodes a protein with the enzymatic activities of bothacetyl-CoA acetyltransferase and 3-hydroxy-3-methylglutaryl-CoA(HMG-CoA) reductase, the first and third proteins in the pathway, andthe mvaS gene encodes second enzyme in the pathway, HMG-CoA synthase.The mvaE gene was amplified from E. faecalis genomic DNA (ATCC700802D-5) with an E. coli ribosome binding site and a spacer in frontusing the following primers:

CF 07-60 (+) Start of mvaE w/ RBS + ATG start codonSacI (SEQ ID NO: 93)5′-GAGACATGAGCTCAGGAGGTAAAAAAACATGAAAACAGTAGTTATTA TTGCF 07-62 (−) Fuse mvaE to mvaS with RBS in between (SEQ ID NO: 94)5′-TTTATCAATCCCAATTGTCATGTTTTTTTACCTCCTTTATTGTTTTC TTAAATC

The mvaS gene was amplified from E. faecalis genomic DNA (ATCC700802D-5) with a RBS and spacer from E. coli in front using thefollowing primers:

CF 07-61 (+) Fuse mvaE to mvaS with RBS in between (SEQ ID NO: 95)5′-GATTTAAGAAAACAATAAAGGAGGTAAAAAAACATGACAATTGGGA TTGATAAACF 07-102 (−) End of mvaS gene BglII (SEQ ID NO: 96)5′-GACATGACATAGATCTTTAGTTTCGATAAGAACGAACGGT

The PCR fragments were fused together with PCR using the followingprimers:

CF 07-60 (+) Start of mvaE w/ RBS + ATG start codon SacI (SEQ ID NO: 93)5′-GAGACATGAGCTCAGGAGGTAAAAAAACATGAAAACAGTAGTTATT ATTGCF 07-102 (−) End of mvaS gene BglII (SEQ ID NO: 96)5′-GACATGACATAGATCTTTAGTTTCGATAAGAACGAACGGT

The fusion PCR fragment was purified using a Qiagen kit and digestedwith the restriction enzymes Sad and BglII. This digested DNA fragmentwas gel purified using a Qiagen kit and ligated into the commerciallyavailable vector pTrcHis2A, which had been digested with Sad and BgiIIand gel purified.

The ligation mix was transformed into E. coli Top 10 cells and colonieswere selected on LA+50 μg/ml carbenicillin plates. A total of sixcolonies were chosen and grown overnight in LB+50 μg/ml carbenicillinand plasmids were isolated using a Qiagen kit. The plasmids weredigested with Sad and BgiII to check for inserts and one correct plasmidwas sequenced with the following primers:

CF 07-58 (+) Start of mvaE gene (SEQ ID NO: 97)5′-ATGAAAACAGTAGTTATTATTGATGC CF 07-59 (−) End of mvaE gene(SEQ ID NO: 98) 5′-ATGTTATTGTTTTCTTAAATCATTTAAAATAGCCF 07-82 (+) Start of mvaS gene (SEQ ID NO: 99)5′-ATGACAATTGGGATTGATAAAATTAG CF 07-83 (−) End of mvaS gene(SEQ ID NO: 100) 5′-TTAGTTTCGATAAGAACGAACGGTCF 07-86 (+) Sequence in mvaE (SEQ ID NO: 101)5′-GAAATAGCCCCATTAGAAGTATC CF 07-87 (+) Sequence in mvaE(SEQ ID NO: 102) 5′-TTGCCAATCATATGATTGAAAATCCF 07-88 (+) Sequence in mvaE (SEQ ID NO: 103)5′-GCTATGCTTCATTAGATCCTTATCG CF 07-89 (+) Sequence mvaS (SEQ ID NO: 104)5′-GAAACCTACATCCAATCTTTTGCCC

The plasmid called pTrcHis2AUpperPathway#1 was correct by sequencing andwas transformed into the commercially available E. coli strain BL21.Selection was done on LA+50 μg/ml carbenicillin. Two transformants werechosen and grown in LB+50 μg/ml carbenicillin until they reached anOD₆₀₀ of 1.5. Both strains were frozen in a vial at −80° C. in thepresence of glycerol. Strains were designated CF 449 forpTrcHis2AUpperPathway#1 in BL21, isolate #1 and CF 450 forpTrcHis2AUpperPathway#1 in BL21, isolate #2. Both clones were found tobehave identically when analyzed.

V. Cloning of UpperMVA Pathway into pCL1920

The plasmid pTrcHis2AUpperPathway was digested with the restrictionendonuclease Sspl to release a fragment containing pTrc-mvaE-mvaS-(Histag)-terminator. In this fragment, the his-tag was not translated. Thisblunt ended 4.5 kbp fragment was purified from a 1.2% E-gel using theQiagen Gel Purification kit. A dephosphorylated, blunt ended 4.2 kbpfragment from pCL1920 was prepared by digesting the vector with therestriction endonuclease PvuII, treating with SAP and gel purifying froma 1.2% E-gel using the Qiagen Gel Purification kit. The two fragmentswere ligated using the Roche Quick Ligation Kit and transformed intoTOP10 chemically competent cells. Transformants were selected on LAcontaining spectinomycin (50 μg/ml). A correct colony was identified byscreening for the presence of the insert by PCR. The plasmid wasdesignated pCL PtrcUpperPathway (FIGS. 26 and 27A-27D; SEQ ID NO:12).

VI. Strains Expressing the Combined Upper and Lower Mevalonic AcidPathways

To obtain a strain with a complete mevalonic acid pathway plus kudzuisoprene synthase, plasmids pTrcKKDyIkISkan and pCLpTrcUpperPathway wereboth transformed into BL21(λDE3) competent cells (Invitrogen) andtransformants were selected on LA containing kanamycin (50 μg/ml) andSpectinomycin (50 μg/ml). The transformants were checked by plasmid prepto ensure that both plasmids were retained in the host. The strain wasdesignated MCM127.

VII. Production of Mevalonic Acid from Glucose in E. coli/pUpperpathway

Single colonies of the BL21/pTrcHis2A-mvaE/mvaS or FM5/ppTrcHis2A-mvaE/mvaS are inoculated into LB+carbenicillin (100 μg/ml) andare grown overnight at 37° C. with shaking at 200 rpm. These cultureswere diluted into 50 ml medium in 250 ml baffled flasks to an OD₆₀₀ of0.1. The medium was TM3+1 or 2% glucose+carbenicillin (100 μg/ml) orTM3+1% glucose+hydrolyzed soy oil+carbenicillin (100 μg/ml) orTM3+biomass (prepared bagasse, corn stover or switchgrass). Cultureswere grown at 30° C. with shaking at 200 rpm for approximately 2-3 hoursuntil an OD₆₀₀ of 0.4 was reached. At this point the expression from themvaE mvaS construct was induced by the addition of IPTG (400 WVI).Cultures were incubated for a further 20 or 40 hours with samples takenat 2 hour intervals to 6 hour post induction and then at 24, 36 and 48hours as needed. Sampling was done by removing 1 ml of culture,measuring the OD₆₀₀, pelleting the cells in a microfuge, removing thesupernatant and analyzing it for mevalonic acid.

A 14 liter fermentation of E. coli cells with nucleic acids encodingEnterococcus faecalis AA-CoA thiolase, HMG-CoA synthase, and HMG-CoAreductase polypeptides produced 22 grams of mevalonic acid with TM3medium and 2% glucose as the cell medium. A shake flask of these cellsproduced 2-4 grams of mevalonic acid per liter with LB medium and 1%glucose as the cell culture medium. The production of mevalonic acid inthese strains indicated that the MVA pathway was functional in E. coli.

VIII. Production of Isoprene from E. coli BL21 Containing the Upper andLower MVA Pathway Plus Kudzu Isoprene Synthase.

The following strains were created by transforming in variouscombinations of plasmids containing the upper and lower MVA pathway andthe kudzu isoprene synthase gene as described above and the plasmidscontaining the idi, dxs, and dxr and isoprene synthase genes describedin Example 7. The host cells used were chemically competent BL21(λDE3)and the transformations were done by standard methods. Transformantswere selected on L agar containing kanamycin (50 μg/ml) or kanamycinplus spectinomycin (both at a concentration of 50 μg/ml). Plates weregrown at 37° C. The resulting strains were designated as follows:

Grown on Kanamycin plus Spectinomycin (50 μg/ml each)MCM127-pCL Upper MVA+pTrcKKDyIkIS (kan) in BL21(λDE3)MCM131-pCL1920+pTrcKKDyIkIS (kan) in BL21(λDE3)MCM125-pCL Upper MVA+pTrcHis2B (kan) in BL21(λDE3)Grown on Kanamycin (50 μg/ml)MCM64-pTrcKudzu yIDI DXS (kan) in BL21(λDE3)MCM50-pTrcKudzu (kan) in BL21(λDE3)MCM123-pTrcKudzu yIDI DXS DXR (kan) in BL21(λDE3)

The above strains were streaked from freezer stocks to LA+appropriateantibiotic and grown overnight at 37° C. A single colony from each platewas used to inoculate shake flasks (25 ml LB+the appropriateantibiotic). The flasks were incubated at 22° C. overnight with shakingat 200 rpm. The next morning the flasks were transferred to a 37° C.incubator and grown for a further 4.5 hours with shaking at 200 rpm. The25 ml cultures were centrifuged to pellet the cells and the cells wereresuspended in 5 ml LB+the appropriate antibiotic. The cultures werethen diluted into 25 ml LB+1% glucose+the appropriate antibiotic to anOD₆₀₀ of 0.1. Two flasks for each strain were set up, one set forinduction with IPTG (800 WVI) the second set was not induced. Thecultures were incubated at 37° C. with shaking at 250 rpm. One set ofthe cultures were induced after 1.50 hours (immediately followingsampling time point 1). At each sampling time point, the OD₆₀₀ wasmeasured and the amount of isoprene determined as described inExample 1. Results are presented in Table 3. The amount of isoprene madeis presented as the amount at the peak production for the particularstrain.

TABLE 3 Production of isoprene in E. coli strains Strain Isoprene(μg/liter/OD/hr) MCM50   23.8 MCM64 289 MCM125 ND MCM131 Trace MCM127874 ND: not detected Trace: peak present but not integratable.

IX. Analysis of Mevalonic Acid

Mevalonolactone (1.0 g, 7.7 mmol) (CAS#503-48-0) was supplied fromSigma-Aldrich (WI, USA) as a syrup that was dissolved in water (7.7 mL)and was treated with potassium hydroxide (7.7 mmol) in order to generatethe potassium salt of mevalonic acid. The conversion to mevalonic acidwas confirmed by ¹H NMR analysis. Samples for HPLC analysis wereprepared by centrifugation at 14,000 rpm for 5 minutes to remove cells,followed by the addition of a 300 μl aliquot of supernatant to 900 μl ofH₂O. Perchloric acid (36 μl of a 70% solution) was then added followedby mixing and cooling on ice for 5 minutes. The samples were thencentrifuged again (14,000 rpm for 5 min) and the supernatant transferredto HPLC. Mevalonic acid standards (20, 10, 5, 1 and 0.5 g/L) wereprepared in the same fashion. Analysis of mevalonic acid (20 μLinjection volume) was performed by HPLC using a BioRad Aminex 87-H+column (300 mm by 7.0 mm) eluted with 5 mM sulfuric acid at 0.6 mL/minwith refractive index (R1) detection. Under these conditions mevalonicacid eluted as the lactone form at 18.5 minutes.

X. Production of Isoprene from E. coli BL21 Containing the Upper MVAPathway Plus Kudzu Isoprene Synthase

A 15-L scale fermentation of E. coli expressing mevalonic acid pathwaypolypeptides and Kudzu isoprene synthase was used to produce isoprenefrom cells in fed-batch culture. This experiment demonstrates thatgrowing cells under glucose limiting conditions resulted in theproduction of 2.2 g/L of isoprene.

Medium Recipe (Per Liter Fermentation Medium):

The medium was generated using the following components per literfermentation medium: K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2 g, citric acidmonohydrate 2 g, ferric ammonium citrate 0.3 g, yeast extract 0.5 g, and1000× modified trace metal solution 1 ml. All of the components wereadded together and dissolved in diH₂O. This solution was autoclaved. ThepH was adjusted to 7.0 with ammonium hydroxide (30%) and q.s. to volume.Glucose 10 g, thiamine*HCl 0.1 g, and antibiotics were added aftersterilization and pH adjustment.

1000× Modified Trace Metal Solution:

The 1000× modified trace metal solution was generated using thefollowing components: citric acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g,FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃100 mg, and NaMoO₄*2H₂O 100 mg. Each component was dissolved one at atime in diH₂O, pH to 3.0 with HCl/NaOH, then q.s. to volume, and filtersterilized with a 0.22 micron filter.

Fermentation was performed in a 15-L bioreactor with BL21 (DE3) E. colicells containing the pCL PtrcUpperPathway (FIG. 26) and pTrcKKDyIkISplasmids. This experiment was carried out to monitor isoprene formationfrom glucose at the desired fermentation pH 7.0 and temperature 30° C.An inoculum of E. coli strain taken from a frozen vial was streaked ontoan LB broth agar plate (with antibiotics) and incubated at 37° C. Asingle colony was inoculated into soytone-yeast extract-glucose medium.After the inoculum grew to OD 1.0 when measured at 550 nm, 500 mL wasused to inoculate a 5-L bioreactor.

Glucose was fed at an exponential rate until cells reached thestationary phase. After this time the glucose feed was decreased to meetmetabolic demands. The total amount of glucose delivered to thebioreactor during the 54 hour fermentation was 3.7 kg. Induction wasachieved by adding isopropyl-beta-D-1-thiogalactopyranoside (IPTG). TheIPTG concentration was brought to 25 μM when the optical density at 550nm (OD₅₅₀) reached a value of 10. The IPTG concentration was raised to50 μM when OD₅₅₀ reached 190. IPTG concentration was raised to 100 μM at38 hours of fermentation. The OD₅₅₀ profile within the bioreactor overtime is shown in FIG. 54. The isoprene level in the off gas from thebioreactor was determined as described herein. The isoprene titerincreased over the course of the fermentation to a final value of 2.2g/L (FIG. 55). The total amount of isoprene produced during the 54 hourfermentation was 15.9 g, and the time course of production is shown inFIG. 56.

XI. Isoprene Fermentation from E. coli Expressing Genes from theMevalonic Acid Pathway and Grown in Fed-Batch Culture at the 15-L Scale

A 15-L scale fermentation of E. coli expressing mevalonic acid pathwaypolypeptides and Kudzu isoprene synthase was used to produce isoprenefrom cells in fed-batch culture. This experiment demonstrates thatgrowing cells under glucose limiting conditions resulted in theproduction of 3.0 g/L of isoprene.

Medium Recipe (Per Liter Fermentation Medium):

The medium was generated using the following components per literfermentation medium: K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2 g, citric acidmonohydrate 2 g, ferric ammonium citrate 0.3 g, yeast extract 0.5 g, and1000× Modified Trace Metal Solution 1 ml. All of the components wereadded together and dissolved in diH₂O. This solution was autoclaved. ThepH was adjusted to 7.0 with ammonium hydroxide (30%) and q.s. to volume.Glucose 10 g, thiamine*HCl 0.1 g, and antibiotics were added aftersterilization and pH adjustment.

1000× Modified Trace Metal Solution:

The 1000× modified trace metal solution was generated using thefollowing components: citric acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g,FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃100 mg, and NaMoO₄*2H₂O 100 mg. Each component was dissolved one at atime in diH₂O, pH to 3.0 with HCl/NaOH, then q.s. to volume, and filtersterilized with a 0.22 micron filter.

Fermentation was performed in a 15-L bioreactor with BL21 (DE3) E. colicells containing the pCL PtrcUpperMVA and pTrc KKDyIkIS plasmids. Thisexperiment was carried out to monitor isoprene formation from glucose atthe desired fermentation pH 7.0 and temperature 30° C. An inoculum of E.coli strain taken from a frozen vial was streaked onto an LB broth agarplate (with antibiotics) and incubated at 37° C. A single colony wasinoculated into tryptone-yeast extract medium. After the inoculum grewto OD 1.0, measured at 550 nm, 500 mL was used to inoculate a 5-Lbioreactor.

Glucose was fed at an exponential rate until cells reached thestationary phase. After this time, the glucose feed was decreased tomeet metabolic demands. The total amount of glucose delivered to thebioreactor during the 59 hour fermentation was 2.2 kg. Induction wasachieved by adding IPTG. The IPTG concentration was brought to 25 μMwhen the optical density at 550 nm (OD₅₅₀) reached a value of 10. TheIPTG concentration was raised to 50 μM when OD₅₅₀ reached 190. The OD₅₅₀profile within the bioreactor over time is shown in FIG. 93. Theisoprene level in the off gas from the bioreactor was determined asdescribed herein. The isoprene titer increased over the course of thefermentation to a final value of 3.0 g/L (FIG. 94). The total amount ofisoprene produced during the 59 hour fermentation was 22.8 g, and thetime course of production is shown in FIG. 95. The molar yield ofutilized carbon that went into producing isoprene during fermentationwas 2.2%. The weight percent yield of isoprene from glucose was 1.0%.

XII. Isoprene Fermentation from E. coli Expressing Genes from theMevalonic Acid Pathway and Grown in Fed-Batch Culture at the 15-L Scale

A 15-L scale fermentation of E. coli expressing mevalonic acid pathwaypolypeptides, Pueraria lobata isoprene synthase, and Kudzu isoprenesynthase was used to produce isoprene from cells in fed-batch culture.This experiment demonstrates that growing cells under glucose limitingconditions resulted in the production of 3.3 g/L of isoprene.

i) Construction of pCLPtrcUpperPathwayHGS2

The gene encoding isoprene synthase from Pueraria lobata wasPCR-amplified using primers NsiI-RBS-HGS F(CTTGATGCATCCTGCATTCGCCCTTAGGAGG, SEQ ID NO:105) andpTrcR(CCAGGCAAATTCTGTTTTATCAG, SEQ ID NO:106), and pTrcKKDyIkIS as atemplate. The PCR product thus obtained was restriction-digested withNsiI and PstI and gel-purified. The plasmid pCL PtrcUpperPathway wasrestriction-digested with PstI and dephosphorylated using rAPid alkalinephosphatase (Roche) according to manufacturer's instructions.

These DNA fragments were ligated together and the ligation reaction wastransformed into E. coli Top10 chemcially competent cells (Invitrogen),plated on L agar containing spectinomycin (50 μg/ml) and incubatedovernight at 37° C. Plasmid DNA was prepared from 6 clones using theQiaquick Spin Mini-prep kit. The plasmid DNA was digested withrestriction enzymes EcoRV and MiuI to identify a clone in which theinsert had the right orientation (i.e., the gene oriented in the sameway as the pTrc promoter).

The resulting correct plasmid was designated pCLPtrcUpperPathwayHGS2.This plasmid was assayed using the headspace assay described herein andfound to produce isoprene in E. coli Top10, thus validating thefunctionality of the gene. The plasmid was transformed into BL21(LDE3)containing pTrcKKDyIkIS to yield the strainBL21/pCLPtrcUpperPathwayHGS2-pTrcKKDyIkIS. This strain has an extra copyof the isoprene synthase compared to the BL21/pCL PtrcUpperMVA and pTrcKKDyIkIS strain (Example 8, part XI). This strain also had increasedexpression and activity of HMGS compared to the BL21/pCL PtrcUpperMVAand pTrc KKDyIkIS strain used in Example 8, part XI.

ii) Isoprene Fermentation from E. coli ExpressingpCLPtrcUpperPathwayHGS2-pTrcKKDyIkIS and Grown in Fed-Batch Culture atthe 15-L Scale

Medium Recipe (per liter fermentation medium):

The medium was generated using the following components per literfermentation medium: K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2 g, citric acidmonohydrate 2 g, ferric ammonium citrate 0.3 g, yeast extract 0.5 g, and1000× modified trace metal solution 1 ml. All of the components wereadded together and dissolved in diH₂O. This solution was autoclaved. ThepH was adjusted to 7.0 with ammonium hydroxide (30%) and q.s. to volume.Glucose 10 g, thiamine*HCl 0.1 g, and antibiotics were added aftersterilization and pH adjustment.

1000× Modified Trace Metal Solution:

The 1000× modified trace metal solution was generated using thefollowing components: citric acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g,FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃100 mg, and NaMoO₄*2H₂O 100 mg. Each component is dissolved one at atime in Di H₂O, pH to 3.0 with HCl/NaOH, then q.s. to volume and filtersterilized with 0.22 micron filter.

Fermentation was performed in a 15-L bioreactor with BL21 (DE3) E. colicells containing the pCLPtrcUpperPathwayHGS2 and pTrc KKDyIkIS plasmids.This experiment was carried out to monitor isoprene formation fromglucose at the desired fermentation pH 7.0 and temperature 30° C. Aninoculum of E. coli strain taken from a frozen vial was streaked onto anLB broth agar plate (with antibiotics) and incubated at 37° C. A singlecolony was inoculated into tryptone-yeast extract medium. After theinoculum grew to OD 1.0 measured at 550 nm, 500 mL was used to inoculatea 5-L bioreactor.

Glucose was fed at an exponential rate until cells reached thestationary phase. After this time the glucose feed was decreased to meetmetabolic demands. The total amount of glucose delivered to thebioreactor during the 58 hour fermentation was 2.1 kg. Induction wasachieved by adding IPTG. The IPTG concentration was brought to 25 μMwhen the optical density at 550 nm (OD₅₅₀) reached a value of 9. TheIPTG concentration was raised to 50 μM when OD₅₅₀ reached 170. The OD₅₅₀profile within the bioreactor over time is shown in FIG. 104. Theisoprene level in the off gas from the bioreactor was determined asdescribed herein. The isoprene titer increased over the course of thefermentation to a final value of 3.3 g/L (FIG. 105). The total amount ofisoprene produced during the 58 hour fermentation was 24.5 g and thetime course of production is shown in FIG. 106. The molar yield ofutilized carbon that went into producing isoprene during fermentationwas 2.5%. The weight percent yield of isoprene from glucose was 1.2%.Analysis showed that the activity of the isoprene synthase was increasedby approximately 3-4 times that compared to BL21 expressing CLPtrcUpperMVA and pTrc KKDyIkIS plasmids (data not shown).

XIII. Chromosomal Integration of the Lower Mevalonate Pathway in E.coli.

A synthetic operon containing mevalonate kinase, mevalonate phosphatekinase, mevalonate pyrophosphate decarboxylase, and the IPP isomerasewas integrated into the chromosome of E. coli. If desired, expressionmay be altered by integrating different promoters 5′ of the operon.

Table 4 lists primers used for this experiment.

TABLE 4 Primers MCM78 attTn7 up rev forgcatgctcgagcggccgcTTTTAATCAAACATCCTGCCAA integration constructCTC (SEQ ID NO: 107) MCM79 attTn7 down rev forgatcgaagggcgatcgTGTCACAGTCTGGCGAAACCG integration construct(SEQ ID NO: 108) MCM88 attTn7 up forw forctgaattctgcagatatcTGTTTTTCCACTCTTCGTTCACTT integration constructT (SEQ ID NO: 109) MCM89 attTn7 down forw fortctagagggcccAAGAAAAATGCCCCGCTTACG (SEQ integration construct ID NO: 110)MCM104 GI1.2 promoter -Gatcgcggccgcgcccttgacgatgccacatcctgagcaaataattcaaccac MVKtaattgtgagcggataacacaaggaggaaacagctatgtcattaccgttcttaacttc (SEQ ID NO: 111) MCM105 aspA terminator -Gatcgggccccaagaaaaaaggcacgtcatctgacgtgccttttttatttgtaga yIDIcgcgttgttatagcattcta (SEQ ID NO: 112) MCM120 Forward of attTn7:aaagtagccgaagatgacggtttgtcacatggagttggcaggatgtttgattaaaattTn7 homology, GB agcAATTAACCCTCACTAAAGGGCGG (SEQ ID marker homologyNO: 113) MCM127 Rev complement of AGAGTGTTCACCAAAAATAATAACCTTTCCCGG1.2 GI: GB marker TGCAgaagttaagaacggtaatgacatagctgtttcctccttgtgttatccgcthomology(extra long),cacaattagtggttgaattatttgctcaggatgtggcatcgtcaagggcTAAT promoter, RBS, ATGACGACTCACTATAGGGCTCG (SEQ ID NO: 114)

i) Target Vector Construction

The attTn7 site was selected for integration. Regions of homologyupstream (attTn7 up) (primers MCM78 and MCM79) and downstream (attTn7down) (primers MCM88 and MCM89) were amplified by PCR from MG1655 cells.A 50 μL reaction with 1 μL 10 μM primers, 3 μL ddH₂O, 45 μL InvitrogenPlatinum PCR Supermix High Fidelity, and a scraped colony of MG1655 wasdenatured for 2:00 at 94° C., cycled 25 times (2:00 at 94° C., 0:30 at50° C., and 1:00 at 68° C.), extended for 7:00 at 72° C., and cooled to4° C. This resulting DNA was cloned into pCR2.1 (Invitrogen) accordingto the manufacturer's instructions, resulting in plasmids MCM278 (attTn7up) and MCM252 (attTn7 down). The 832 bp ApaI-Pvul fragment digested andgel purified from MCM252 was cloned into ApaI-PvuI digested and gelpurified plasmid pR6K, creating plasmid MCM276. The 825 bp PstI-NotIfragment digested and gel purified from MCM278 was cloned into PstI-NotIdigested and gel purified MCM276, creating plasmid MCM281.

ii) Cloning of Lower Pathway and Promoter

MVK-PMK-MVD-IDI genes were amplified from pTrcKKDyIkIS with primersMCM104 and MCM105 using Roche Expand Long PCR System according to themanufacturer's instructions. This product was digested with NotI andApaI and cloned into MCM281 which had been digested with NotI and ApaIand gel purified. Primers MCM120 and MCM127 were used to amplify CMRcassette from the GeneBridges FRT-gb2-Cm-FRT template DNA usingStratagene Pfu Ultra II. A PCR program of denaturing at 95° C. for 4:00,5 cycles of 95° C. for 0:20, 55° C. for 0:20, 72° C. for 2:00, 25 cyclesof 95° C. for 0:20, 58° C. for 0:20, 72° C. for 2:00, 72° C. for 10:00,and then cooling to 4° C. was used with four 50 μL PCR reactionscontaining 1 μL˜10 ng/μL template, 1 μL each primer, 1.25 μL 10 mMdNTPs, 5 μL 10× buffer, 1 μL enzyme, and 39.75 μL ddH₂O. Reactions werepooled, purified on a Qiagen PCR cleanup column, and used toelectroporate water-washed Pir1 cells containing plasmid MCM296.Electroporation was carried out in 2 mM cuvettes at 2.5V and 200 ohms.Electroporation reactions were recovered in LB for 3 hr at 30° C.Transformant MCM330 was selected on LA with CMP5, Kan50 (FIGS. 107 and108A-108C; SEQ ID NO:25).

iii) Integration into E. coli Chromosome

Miniprepped DNA (Qiaquick Spin kit) from MCM330 was digested with SnaBIand used to electroporate BL21(DE3) (Novagen) or MG1655 containingGeneBridges plasmid pRedET Carb. Cells were grown at 30° C. to ˜OD1 theninduced with 0.4% L-arabinose at 37° C. for 1.5 hours. These cells werewashed three times in 4° C. ddH2O before electroporation with 2 μL ofDNA. Integrants were selected on L agar with containing chloramphenicol(5 μg/ml) and subsequently confirmed to not grow on L agar+Kanamycin (50μg/ml). BL21 integrant MCM331 and MG1655 integrant MCM333 were frozen.

iv) Construction of pET24D-Kudzu encoding Kudzu Isoprene Synthase

The kudzu isoprene synthase gene was subcloned into the pET24d vector(Novagen) from the pCR2.1 vector (Invitrogen). In particular, the kudzuisoprene synthase gene was amplified from the pTrcKudzu template DNAusing primers MCM50 5′-GATCATGCAT TCGCCCTTAG GAGGTAAAAA AACATGTGTGCGACCTCTTC TCAATTTACT (SEQ ID NO:52) and MCM53 5′-CGGTCGACGG ATCCCTGCAGTTAGACATAC ATCAGCTG (SEQ ID NO:50). PCR reactions were carried out usingTaq DNA Polymerase (Invitrogen), and the resulting PCR product wascloned into pCR2.1-TOPO TA cloning vector (Invitrogen), and transformedinto E. coli Top10 chemically competent cells (Invitrogen).Transformants were plated on L agar containing carbenicillin (50 μg/ml)and incubated overnight at 37° C. Five ml Luria Broth culturescontaining carbenicillin 50 μg/ml were inoculated with singletransformants and grown overnight at 37° C. Five colonies were screenedfor the correct insert by sequencing of plasmid DNA isolated from 1 mlof liquid culture (Luria Broth) and purified using the QIAprep SpinMini-prep Kit (Qiagen). The resulting plasmid, designated MCM93,contains the kudzu isoprene synthase coding sequence in a pCR2.1backbone.

The kudzu coding sequence was removed by restriction endonucleasedigestion with PciI and BamH1 (Roche) and gel purified using theQIAquick Gel Extraction kit (Qiagen). The pET24d vector DNA was digestedwith NcoI and BamHI (Roche), treated with shrimp alkaline phosphatase(Roche), and purified using the QIAprep Spin Mini-prep Kit (Qiagen). Thekudzu isoprene synthase fragment was ligated to the NcoI/BamH1 digestedpET24d using the Rapid DNA Ligation Kit (Roche) at a 5:1 fragment tovector ratio in a total volume of 20 μl. A portion of the ligationmixture (5 μl) was transformed into E. coli Top 10 chemically competentcells and plated on L agar containing kanamycin (50 μg/ml). The correcttransformant was confirmed by sequencing and transformed into chemicallycompetent BL21(λDE3)pLysS cells (Novagen). A single colony was selectedafter overnight growth at 37° C. on L agar containing kanamycin (50μg/ml). A map of the resulting plasmid designated as pET24D-Kudzu isshown in FIG. 109. The sequence of pET24D-Kudzu (SEQ ID NO:26) is shownin FIGS. 110A and 110B. Isoprene synthase activity was confirmed using aheadspace assay.

v) Production Strains

Strains MCM331 and MCM333 were cotransformed with plasmidspCLPtrcupperpathway and either pTrcKudzu or pETKudzu, resulting in thestrains shown in Table 5.

TABLE 5 Production Strains Isoprene Integrated Upper MVA synthaseProduction Background Lower plasmid plasmid Stain BL21(DE3) MCM331pCLPtrcUpper pTrcKudzu MCM343 Pathway BL21(DE3) MCM331 pCLPtrcUpperpET24D- MCM335 Pathway Kudzu MG1655 MCM333 pCLPtrcUpper pTrcKudzu MCM345Pathwayvi) Isoprene Fermentation from E. coli Expressing Genes from theMevalonic Acid Pathway and Grown in Fed-Batch Culture at the 15-L Scale.

Medium Recipe (per liter fermentation medium):

The medium was generated using the following components per literfermentation medium: K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2 g, citric acidmonohydrate 2 g, ferric ammonium citrate 0.3 g, yeast extract 0.5 g, and1000× modified trace metal solution 1 ml. All of the components wereadded together and dissolved in diH2O. This solution was autoclaved. ThepH was adjusted to 7.0 with ammonium hydroxide (30%) and q.s. to volume.Glucose 10 g, thiamine*HCl 0.1 g, and antibiotics were added aftersterilization and pH adjustment.

1000× Modified Trace Metal Solution:

The 1000× modified trace metal solution was generated using thefollowing components: citric acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g,FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃100 mg, and NaMoO₄*2H₂O 100 mg. Each component is dissolved one at atime in Di H₂O, pH to 3.0 with HCl/NaOH, then q.s. to volume and filtersterilized with a 0.22 micron filter.

Fermentation was performed in a 15-L bioreactor with BL21 (DE3) E. colicells containing the gi1.2 integrated lower MVA pathway described aboveand the pCL PtrcUpperMVA and pTrcKudzu plasmids. This experiment wascarried out to monitor isoprene formation from glucose at the desiredfermentation pH 7.0 and temperature 30° C. An inoculum of E. coli straintaken from a frozen vial was streaked onto an LB broth agar plate (withantibiotics) and incubated at 37° C. A single colony was inoculated intotryptone-yeast extract medium. After the inoculum grew to OD 1.0,measured at 550 nm, 500 mL was used to inoculate a 5-L bioreactor.

Glucose was fed at an exponential rate until cells reached thestationary phase. After this time, the glucose feed was decreased tomeet metabolic demands. The total amount of glucose delivered to thebioreactor during the 57 hour fermentation was 3.9 kg. Induction wasachieved by adding IPTG. The IPTG concentration was brought to 100 μMwhen the carbon dioxide evolution rate reached 100 mmol/L/hr. The OD₅₅₀profile within the bioreactor over time is shown in FIG. 111A. Theisoprene level in the off gas from the bioreactor was determined asdescribed herein. The isoprene titer increased over the course of thefermentation to a final value of 1.6 g/L (FIG. 111B). The specificproductivity of isoprene over the course of the fermentation is shown inFIG. 111C and peaked at 1.2 mg/OD/hr. The total amount of isopreneproduced during the 57 hour fermentation was 16.2 g. The molar yield ofutilized carbon that went into producing isoprene during fermentationwas 0.9%. The weight percent yield of isoprene from glucose was 0.4%.

XIV. Production of Isoprene from E. coli BL21 Containing the KudzuIsoprene Synthase Using Glycerol as a Carbon Source

A 15-L scale fermentation of E. coli expressing Kudzu isoprene synthasewas used to produce isoprene from cells fed glycerol in fed-batchculture. This experiment demonstrates that growing cells in the presenceof glycerol (without glucose) resulted in the production of 2.2 mg/L ofisoprene.

Medium Recipe (Per Liter Fermentation Medium):

The medium was generated using the following components per literfermentation medium: K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2 g, citric acidmonohydrate 2 g, ferric ammonium citrate 0.3 g, and 1000× modified tracemetal solution 1 ml. All of the components were added together anddissolved in diH₂O. This solution was autoclaved. The pH was adjusted to7.0 with ammonium hydroxide (30%) and q.s. to volume. Glycerol 5.1 g,thiamine*HCl 0.1 g, and antibiotics were added after sterilization andpH adjustment.

1000× Modified Trace Metal Solution:

The medium was generated using the following components per literfermentation medium: citric acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g,FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃100 mg, and NaMoO₄*2H₂O 100 mg. Each component was dissolved one at atime in diH₂O, pH to 3.0 with HCl/NaOH, then q.s. to volume and filtersterilized with a 0.22 micron filter.

Fermentation was performed in a 15-L bioreactor with BL21 (DE3) E. colicells containing the pTrcKudzu plasmid. This experiment was carried outto monitor isoprene formation from glycerol at the desired fermentationpH 7.0 and temperature 35° C. An inoculum of E. coli strain taken from afrozen vial was streaked onto an LA broth agar plate (with antibiotics)and incubated at 37° C. A single colony was inoculated intosoytone-yeast extract-glucose medium and grown at 35° C. After theinoculum grew to OD 1.0, measured at 550 nm, 600 mL was used toinoculate a 7.5-L bioreactor.

Glycerol was fed at an exponential rate until cells reached an opticaldensity at 550 nm (OD₅₅₀) of 153. The total amount of glycerol deliveredto the bioreactor during the 36 hour fermentation was 1.7 kg. Other thanthe glucose in the inoculum, no glucose was added to the bioreactor.Induction was achieved by adding IPTG. The IPTG concentration wasbrought to 20 μM when the OD₅₅₀ reached a value of 50. The OD₅₅₀ profilewithin the bioreactor over time is shown in FIG. 57. The isoprene levelin the off gas from the bioreactor was determined as described herein.The isoprene titer increased over the course of the fermentation to afinal value of 2.2 mg/L (FIG. 58). The total amount of isoprene producedduring the 54 hour fermentation was 20.9 mg, and the time course ofproduction is shown in FIG. 59.

XV. Isoprene Fermentation from E. coli Expressing Genes from theMevalonic Acid Pathway and Grown in Fed-Batch Culture at the 15-L ScaleUsing Invert Sugar as a Carbon Source

A 15-L scale fermentation of E. coli expressing mevalonic acid pathwaypolypeptides and Kudzu isoprene synthase was used to produce isoprenefrom cells fed invert sugar in fed-batch culture. This experimentdemonstrates that growing cells in the presence of invert sugar resultedin the production of 2.4 g/L of isoprene.

Medium Recipe (Per Liter Fermentation Medium):

The medium was generated using the following components per literfermentation medium: K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2 g, citric acidmonohydrate 2 g, ferric ammonium citrate 0.3 g, yeast extract 0.5 g, and1000× Modified Trace Metal Solution 1 ml. All of the components wereadded together and dissolved in diH₂O. This solution was autoclaved. ThepH was adjusted to 7.0 with ammonium hydroxide (30%) and q.s. to volume.Invert sugar 10 g, thiamine*HCl 0.1 g, and antibiotics were added aftersterilization and pH adjustment.

1000× Modified Trace Metal Solution:

The 1000× modified trace metal solution was generated using thefollowing components: citric acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g,FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃100 mg, and NaMoO₄*2H₂O 100 mg. Each component is dissolved one at atime in Di H2O, pH to 3.0 with HCl/NaOH, then q.s. to volume and filtersterilized with 0.22 micron filter.

Fermentation was performed in a 15-L bioreactor with BL21 (DE3) E. colicells containing the pCL PtrcUpperMVA and pTrc KKDyIkIS plasmids. Thisexperiment was carried out to monitor isoprene formation from invertsugar at the desired fermentation pH 7.0 and temperature 30° C. Aninoculum of E. coli strain taken from a frozen vial was streaked onto anLB broth agar plate (with antibiotics) and incubated at 37° C. A singlecolony was inoculated into tryptone-yeast extract medium. After theinoculum grew to OD 1.0, measured at 550 nm, 500 mL was used toinoculate a 5-L bioreactor.

Invert sugar was fed at an exponential rate until cells reached thestationary phase. After this time the invert sugar feed was decreased tomeet metabolic demands. The total amount of invert sugar delivered tothe bioreactor during the 44 hour fermentation was 2.4 kg. Induction wasachieved by adding IPTG. The IPTG concentration was brought to 25 μMwhen the optical density at 550 nm (OD₅₅₀) reached a value of 9. TheIPTG concentration was raised to 50 μM when OD₅₅₀ reached 200. The OD₅₅₀profile within the bioreactor over time is shown in FIG. 96. Theisoprene level in the off gas from the bioreactor was determined asdescribed herein. The isoprene titer increased over the course of thefermentation to a final value of 2.4 g/L (FIG. 97). The total amount ofisoprene produced during the 44 hour fermentation was 18.4 g and thetime course of production is shown in FIG. 98. The molar yield ofutilized carbon that went into producing isoprene during fermentationwas 1.7%. The weight percent yield of isoprene from glucose was 0.8%.

Example 9 Construction of the Upper and Lower MVA Pathway forIntegration into Bacillus subtilis

I. Construction of the Upper MVA Pathway in Bacillus subtilis

The upper pathway from Enterococcus faecalis is integrated into B.subtilis under control of the aprE promoter. The upper pathway consistsof two genes; mvaE, which encodes for AACT and HMGR, and mvaS, whichencodes for HMGS. The two genes are fused together with a stop codon inbetween, an RBS site in front of mvaS, and are under the control of theaprE promoter. A terminator is situated after the mvaE gene. Thechloramphenicol resistance marker is cloned after the mvaE gene and theconstruct is integrated at the aprE locus by double cross over usingflanking regions of homology.

Four DNA fragments are amplified by PCR such that they contain overhangsthat will allowed them to be fused together by a PCR reaction. PCRamplifications are carried out using Herculase polymerase according tomanufacturer's instructions.

1. PaprE CF 07-134 (+) Start of aprE promoter PstI (SEQ ID NO: 115)5′-GACATCTGCAGCTCCATTTTCTTCTGC CF 07-94 (−) Fuse PaprE to mvaE(SEQ ID NO: 116) 5′-CAATAATAACTACTGTTTTCACTCTTTACCCTCTCCTTTTAATemplate: Bacillus subtilis chromosomal DNA 2. mvaECF 07-93 (+) fuse mvaE to the aprE promoter (GTG start codon)(SEQ ID NO: 117) 5′-TTAAAAGGAGAGGGTAAAGAGTGAAAACAGTAGTTATTATTGCF 07-62 (−) Fuse mvaE to mvaS with RBS in between (SEQ ID NO: 94)5′-TTTATCAATCCCAATTGTCATGTTTTTTTACCTCCTTTATTGTTTTCTTAAATCTemplate: Enterococcus faecalis chromosomal DNA (from ATCC) 3. mvaSCF 07-61 (+) Fuse mvaE to mvaS with RBS in between (SEQ ID NO: 95)5′-GATTTAAGAAAACAATAAAGGAGGTAAAAAAACATGACAATTGGGATTGATAAACF 07-124 (−) Fuse the end of mvaS to the terminator (SEQ ID NO: 118)5′-CGGGGCCAAGGCCGGTTTTTTTTAGTTTCGATAAGAACGAACGGTTemplate: Enterococcus faecalis chromosomal DNA4. B. amyliquefaciens alkaline serine protease terminatorCF 07-123 (+) Fuse the end of mvaS to the terminator (SEQ ID NO: 119)5′-ACCGTTCGTTCTTATCGAAACTAAAAAAAACCGGCCTTGGCCCCGCF 07-46 (−) End of B. amyliquefaciens terminator BamHI (SEQ ID NO: 58)5′-GACATGACGGATCCGATTACGAATGCCGTCTCTemplate: Bacillus amyliquefaciens chromosomal DNA PCR Fusion Reactions5. Fuse mvaE to mvaSCF 07-93 (+) fuse mvaE to the aprE promoter (GTG start codon)(SEQ ID NO: 117) 5′-TTAAAAGGAGAGGGTAAAGAGTGAAAACAGTAGTTATTATTGCF 07-124 (−) Fuse the end of mvaS to the terminator (SEQ ID NO: 118)5′-CGGGGCCAAGGCCGGTTTTTTTTAGTTTCGATAAGAACGAACGGTTemplate: #2 and 3 from above 6. Fuse mvaE-mvaS to aprE promoterCF 07-134 (+) Start of aprE promoter PstI (SEQ ID NO: 115)5′-GACATCTGCAGCTCCATTTTCTTCTGCCF 07-124 (−) Fuse the end of mvaS to the terminator (SEQ ID NO: 118)5′-CGGGGCCAAGGCCGGTTTTTTTTAGTTTCGATAAGAACGAACGGTTemplate #1 and #4 from above 7. Fuse PaprE-mvaE-mvaS to terminatorCF 07-134 (+) Start of aprE promoter PstI (SEQ ID NO: 115)5′-GACATCTGCAGCTCCATTTTCTTCTGCCF 07-46 (−) End of B. amyliquefaciens terminator BamHI (SEQ ID NO: 58)5′-GACATGACGGATCCGATTACGAATGCCGTCTC Template: #4 and #6

The product is digested with restriction endonucleases PstI/BamHI andligated to pJM102 (Perego, M. 1993. Integrational vectors for geneticmanipulation in Bacillus subtilis, p. 615-624. In A. L. Sonenshein, J.A. Hoch, and R. Losick (ed.), Bacillus subtilis and other gram-positivebacteria: biochemistry, physiology, and molecular genetics. AmericanSociety for Microbiology, Washington, D.C.) which is digested withPstI/BamHI. The ligation is transformed into E. coli TOP 10 chemicallycompetent cells and transformants are selected on LA containingcarbenicillin (50 μg/ml). The correct plasmid is identified bysequencing and is designated pJMUpperpathway2 (FIGS. 50 and 51).Purified plasmid DNA is transformed into Bacillus subtilis aprEnprEPxyl-comK and transformants are selected on L agar containingchloramphenicol (5 μg/ml). A correct colony is selected and is platedsequentially on L agar containing chloramphenicol 10, 15 and 25 μg/ml toamplify the number of copies of the cassette containing the upperpathway.

The resulting strain is tested for mevalonic acid production by growingin LB containing 1% glucose and 1%. Cultures are analyzed by GC for theproduction of mevalonic acid.

This strain is used subsequently as a host for the integration of thelower mevalonic acid pathway.

The following primers are used to sequence the various constructs above.

Sequencing Primers:

CF 07-134 (+) Start of aprE promoter PstI (SEQ ID NO: 115)5′-GACATCTGCAGCTCCATTTTCTTCTGC CF 07-58 (+) Start of mvaE gene(SEQ ID NO: 97) 5′-ATGAAAACAGTAGTTATTATTGATGCCF 07-59 (−) End of mvaE gene (SEQ ID NO: 98)5′-ATGTTATTGTTTTCTTAAATCATTTAAAATAGC CF 07-82 (+) Start of mvaS gene(SEQ ID NO: 99) 5′-ATGACAATTGGGATTGATAAAATTAGCF 07-83 (−) End of mvaS gene (SEQ ID NO: 100)5′-TTAGTTTCGATAAGAACGAACGGT CF 07-86 (+) Sequence in mvaE(SEQ ID NO: 101) 5′-GAAATAGCCCCATTAGAAGTATCCF 07-87 (+) Sequence in mvaE (SEQ ID NO: 102)5′-TTGCCAATCATATGATTGAAAATC CF 07-88 (+) Sequence in mvaE(SEQ ID NO: 103) 5′-GCTATGCTTCATTAGATCCTTATCG CF 07-89 (+) Sequence mvaS(SEQ ID NO: 104) 5′-GAAACCTACATCCAATCTTTTGCCC

Transformants are selected on LA containing chloramphenicol at aconcentration of 5 μg/ml. One colony is confirmed to have the correctintegration by sequencing and is plated on LA containing increasingconcentrations of chloramphenicol over several days, to a final level of25 μg/ml. This results in amplification of the cassette containing thegenes of interest. The resulting strain is designated CF 455:pJMupperpathway#1×Bacillus subtilis aprEnprE Pxyl comK (amplified togrow on LA containing chloramphenicol 25 μg/ml).

II. Construction of the Lower MVA Pathway in Bacillus subtilis

The lower MVA pathway, consisting of the genes mvk1, pmk, mpd and idiare combined in a cassette consisting of flanking DNA regions from thenprE region of the B. subtilis chromosome (site of integration), theaprE promoter, and the spectinomycin resistance marker (see FIGS. 28 and29; SEQ ID NO:13). This cassette is synthesized by DNA2.0 and isintegrated into the chromosome of B. subtilis containing the upper MVApathway integrated at the aprE locus. The kudzu isoprene synthase geneis expressed from the replicating plasmid described in Example 4 and istransformed into the strain with both upper and lower pathwaysintegrated.

Example 10 Exemplary Isoprene Compositions and Methods of Making Them I.Compositional Analysis of Fermentation Off-Gas Containing Isoprene

A 14 L scale fermentation was performed with a recombinant E. coli BL21(DE3) strain containing two plasmids (pCL upperMev; pTrcKKDyIkISencoding the full mevalonate pathway for isoprenoid precursorbiosynthesis, an isoprenyl pyrophosphate isomerase from yeast, and anisoprene synthase from Kudzu. Fermentation off-gas from the 14 L tankwas collected into 20 mL headspace vials at around the time of peakisoprene productivity (27.9 hours elapsed fermentation time, “EFT”) andanalyzed by headspace GC/MS for volatile components.

Headspace analysis was performed with an Agilent 6890 GC/MS systemfitted with an Agilent HP-5MS GC/MS column (30 m×250 μm; 0.25 μm filmthickness). A combiPAL autoinjector was used for sampling 500 μLaliquots from 20 mL headspace vials. The GC/MS method utilized helium asthe carrier gas at a flow of 1 mL/min. The injection port was held at250° C. with a split ratio of 50:1. The oven temperature was held at 37°C. for an initial 2 minute period, followed an increase to 237° C. at arate of 25° C./min for a total method time of 10 minutes. The Agilent5793N mass selective detector scanned from m/z 29 to m/z 300. The limitof detection of this system is approximately 0.1 μg/L_(gas) orapproximately 0.1 ppm. If desired, more sensitive equipment with a lowerlimit of detection may be used.

The off-gas consisted of 99.925% (v/v) permanent gases (N₂, CO₂ and O₂),approximately 0.075% isoprene (2-methyl-1,3-butadiene) (−750 ppmv, 2100μg/L) and minor amounts (<50 ppmv) of ethanol, acetone, and two C5prenyl alcohols. The amount of water vapor was not determined but wasestimated to be equal to the equilibrium vapor pressure at 0° C. Thecomposition of the volatile organic fraction was determined byintegration of the area under the peaks in the GC/MS chromatogram (FIGS.86A and 86B) and is listed in Table 6. Calibration curves for ethanoland acetone standards enabled the conversion of GC area to gas phaseconcentration in units of μg/L using standard methods.

TABLE 6 Composition of volatile organic components in fermentationoff-gas. The off-gas was analyzed at the 27.9 hour time point of afermentation using an E. coli BL21 (DE3) strain expressing aheterologous mevalonate pathway, an isoprenyl pyrophosphate isomerasefrom yeast, and an isoprene synthase from Kudzu. Compound RT (min) GCarea Area % Conc. (μg/L) Ethanol 1.669 239005 0.84 62 +/− 6 Acetone1.703 288352 1.02 42 +/− 4 Isoprene (2-methyl- 1.829 27764544 97.81 2000+/− 200 1,3-butadiene) 3-methyl-3-buten-1-ol 3.493 35060 0.12 <103-methyl-2-buten-1-ol 4.116 58153 0.20 <10II. Measurement of Trace Volatile Organic Compounds (VOCs) co-Producedwith Isoprene During Fermentation of a Recombinant E. coli Strain

A 14 L scale fermentation was performed with a recombinant E. coli BL21(DE3) strain containing two plasmids (pCL upperMev; pTrcKKDyIkIS)encoding the full mevalonate pathway for isoprenoid precursorbiosynthesis, an isoprenyl pyrophosphate isomerase from yeast, and anisoprene synthase from Kudzu.

Fermentation off-gas was passed through cooled headspace vials in orderto concentrate and identify trace volatile organic components. Theoff-gas from this fermentation was sampled at a rate of 1 L/min for 10minutes through a 20 mL headspace vial packed with quartz wool (2 g) andcooled to −78° C. with dry ice. The vial was recapped with a fresh vialcap and analyzed by headspace GC/MS for trapped VOCs using theconditions described in Example 10, part I. The ratios of compoundsobserved in FIGS. 87A-87D are a combination of overall level in thefermentation off-gas, the relative vapor pressure at −78° C., and thedetector response of the mass spectrometer. For example, the low levelof isoprene relative to oxygenated volatiles (e.g., acetone and ethanol)is a function of the high volatility of this material such that it doesnot accumulate in the headspace vial at −78° C.

The presence of many of these compounds is unique to isoprenecompositions derived from biological sources. The results are depictedin FIGS. 87A-87D and summarized in Tables 7A and 7B.

TABLE 7A Trace volatiles present in off-gas produced by E. coli BL21(DE3) (pCL upperMev; pTrcKKDyIkIS) following cryo-trapping at −78° C. RTGC Compound (min) Area1 Area %2 Ratio %3 Acetaldehyde 1.542 40198614.841 40.14 Ethanol 1.634 10553620 12.708 105.39 Acetone 1.727 72363238.714 72.26 2-methyl-1,3-butadiene 1.777 10013714 12.058 100.001-propanol 1.987 163574 0.197 1.63 Diacetyl 2.156 221078 0.266 2.212-methyl-3-buten-2-ol 2.316 902735 1.087 9.01 2-methyl-1-propanol 2.451446387 0.538 4.46 3-methyl-1-butanal 2.7 165162 0.199 1.65 1-butanol2.791 231738 0.279 2.31 3-methyl-3-buten-1-ol 3.514 14851860 17.884148.32 3-methyl-1-butanol 3.557 8458483 10.185 84.473-methyl-2-buten-1-ol 4.042 18201341 21.917 181.76 3-methyl-2-butenal4.153 1837273 2.212 18.35 3-methylbutyl acetate 5.197 196136 0.236 1.963-methyl-3-buten-1-yl 5.284 652132 0.785 6.51 acetate 2-heptanone 5.34867224 0.081 0.67 2,5-dimethylpyrazine 5.591 58029 0.070 0.583-methyl-2-buten-1-yl 5.676 1686507 2.031 16.84 acetate6-methyl-5-hepten-2-one 6.307 101797 0.123 1.02 2,4,5-trimethylpyridine6.39 68477 0.082 0.68 2,3,5-trimethylpyrazine 6.485 30420 0.037 0.30(E)-3,7-dimethyl-1,3,6- 6.766 848928 1.022 8.48 octatriene(Z)-3,7-dimethyl-1,3,6- 6.864 448810 0.540 4.48 octatriene3-methyl-2-buten-1-yl 7.294 105356 0.127 1.05 butyrate Citronellal 7.756208092 0.251 2.08 2,3-cycloheptenolpyridine 8.98 1119947 1.349 11.18 1GCarea is the uncorrected area under the peak corresponding to the listedcompound. 2Area % is the peak area expressed as a % relative to thetotal peak area of all compounds. 3Ratio % is the peak area expressed asa % relative to the peak area of 2-methyl-1,3-butadiene.

TABLE 7B Trace volatiles present in off-gas produced by E. coli BL21(DE3) (pCL upperMev; pTrcKKDyIkIS) following cryo-trapping at −196° C.RT GC Ratio Compound (min) Area1 Area %2 %3 Acetaldehyde 1.54 16557100.276 0.33 Methanethiol 1.584 173620 0.029 0.03 Ethanol 1.631 102596801.707 2.03 Acetone 1.722 73089100 12.164 14.43 2-methyl-1,3-butadiene1.771 506349429 84.269 100.00 methyl acetate 1.852 320112 0.053 0.061-propanol 1.983 156752 0.026 0.03 Diacetyl 2.148 67635 0.011 0.012-butanone 2.216 254364 0.042 0.05 2-methyl-3-buten-2-ol 2.312 6847080.114 0.14 ethyl acetate 2.345 2226391 0.371 0.44 2-methyl-1-propanol2.451 187719 0.031 0.04 3-methyl-1-butanal 2.696 115723 0.019 0.023-methyl-2-butanone 2.751 116861 0.019 0.02 1-butanol 2.792 54555 0.0090.01 2-pentanone 3.034 66520 0.011 0.01 3-methyl-3-buten-1-ol 3.5161123520 0.187 0.22 3-methyl-1-butanol 3.561 572836 0.095 0.11 ethylisobutyrate 3.861 142056 0.024 0.03 3-methyl-2-buten-1-ol 4.048 3025580.050 0.06 3-methyl-2-butenal 4.152 585690 0.097 0.12 butyl acetate4.502 29665 0.005 0.01 3-methylbutyl acetate 5.194 271797 0.045 0.053-methyl-3-buten-1-yl acetate 5.281 705366 0.117 0.143-methyl-2-buten-1-yl acetate 5.675 815186 0.136 0.16(E)-3,7-dimethyl-1,3,6- 6.766 207061 0.034 0.04 octatriene(Z)-3,7-dimethyl-1,3,6- 6.863 94294 0.016 0.02 octatriene2,3-cycloheptenolpyridine 8.983 135104 0.022 0.03 1GC area is theuncorrected area under the peak corresponding to the listed compound.2Area % is the peak area expressed as a % relative to the total peakarea of all compounds. 3Ratio % is the peak area expressed as a %relative to the peak area of 2-methyl-1,3-butadiene.III. Absence of C5 Hydrocarbon Isomers in Isoprene Derived fromFermentation.

Cryo-trapping of isoprene present in fermentation off-gas was performedusing a 2 mL headspace vial cooled in liquid nitrogen. The off-gas (1L/min) was first passed through a 20 mL vial containing sodium hydroxidepellets in order to minimize the accumulation of ice and solid CO₂ inthe 2 mL vial (−196° C.). Approximately 10 L of off-gas was passedthrough the vial, after which it was allowed to warm to −78° C. withventing, followed by resealing with a fresh vial cap and analysis byGC/MS.

GC/MS headspace analysis was performed with an Agilent 6890 GC/MS systemusing a 100 μL gas tight syringe in headspace mode. A Zebron ZB-624GC/MS column (30 m×250 μm; 1.40 μm film thickness) was used forseparation of analytes. The GC autoinjector was fitted with a gas-tight100 μL syringe, and the needle height was adjusted to allow theinjection of a 50 μL headspace sample from a 2 mL GC vial. The GC/MSmethod utilized helium as the carrier gas at a flow of 1 mL/min. Theinjection port was held at 200° C. with a split ratio of 20:1. The oventemperature was held at 37° C. for the 5 minute duration of theanalysis. The Agilent 5793N mass selective detector was run in singleion monitoring (SIM) mode on m/z 55, 66, 67 and 70. Under theseconditions, isoprene was observed to elute at 2.966 minutes (FIG. 88B).A standard of petroleum derived isoprene (Sigma-Aldrich) was alsoanalyzed using this method and was found to contain additional C5hydrocarbon isomers, which eluted shortly before or after the main peakand were quantified based on corrected GC area (FIG. 88A).

TABLE 8A GC/MS analysis of petroleum-derived isoprene Area % of total RTC5 Compound (min) GC area hydrocarbons 2-methyl-1-butene 2.689 18.2 ×103 0.017% (Z)-2-pentene 2.835 10.6 × 104 0.101% Isoprene 2.966 10.4 ×107 99.869% 1,3- 3.297 12.8 × 103 0.012% cyclopentadiene (CPD)

TABLE 8B GC/MS analysis of fermentation-derived isoprene (% total C5hydrocarbons) Corrected GC % of total C5 Compound RT (min) Areahydrocarbons Isoprene 2.966 8.1 × 107 100%

In a separate experiment, a standard mixture of C5 hydrocarbons wasanalyzed to determine if the detector response was the same for each ofthe compounds. The compounds were 2-methyl-1-butene,2-methyl-1,3-butadiene, (E)-2-pentene, (Z)-2-pentene and(E)-1,3-pentadiene. In this case, the analysis was performed on anAgilent DB-Petro column (100 m×0.25 mm, 0.50 μm film thickness) held at50° C. for 15 minutes. The GC/MS method utilized helium as the carriergas at a flow of 1 mL/min. The injection port was held at 200° C. with asplit ratio of 50:1. The Agilent 5793N mass selective detector was runin full scan mode from m/z 19 to m/z 250. Under these conditions, a 100μg/L concentration of each standard produced the same detector responsewithin experimental error.

IV. Compositions Comprising Isoprene Adsorbed to a Solid Phase.

Biologically-produced isoprene was adsorbed to activated carbonresulting in a solid phase containing 50 to 99.9% carbon, 0.1% to 50%isoprene, 0.01% to 5% water, and minor amounts (<0.1%) of other volatileorganic components.

Fermentation off-gas was run through a copper condensation coil held at0° C., followed by a granulated silica desiccant filter in order toremove water vapor. The dehumidified off-gas was then run through carboncontaining filters (Koby Jr, Koby Filters, MA) to the point at whichbreakthrough of isoprene was detected in the filter exhaust by GC/MS.The amount of isoprene adsorbed to the cartridge can be determinedindirectly by calculating the concentration in the off-gas, the overallflow rate and the percent breakthrough over the collection period.Alternately the adsorbed isoprene can be recovered from the filters bythermal, vacuum, or solvent-mediated desorption.

V. Collection and Analysis of Condensed Isoprene.

Fermentation off-gas is dehumidified, and the CO₂ removed by filtrationthrough a suitable adsorbent (e.g., ascarite). The resulting off-gasstream is then run through a liquid nitrogen-cooled condenser in orderto condense the VOCs in the stream. The collection vessel containst-butyl catechol to inhibit the resulting isoprene condensate. Thecondensate is analyzed by GC/MS and NMR in order to determine purityusing standard methods, such as those described herein.

VI. Production of Prenyl Alcohols by Fermentation

Analysis of off-gas from an E. coli BL21 (DE3) strain expressing a Kudzuisoprene synthase revealed the presence of both isoprene and3-methyl-3-buten-1-ol (isoprenol). The levels of the two compounds inthe fermentation off-gas over the fermentation are shown in FIG. 89 asdetermined by headspace GC/MS. Levels of isoprenol(3-methyl-3-buten-1-ol, 3-MBA) attained was nearly 10 μg/L_(offgas) inthis experiment. Additional experiments produced levels of approximately20 μg/L_(offgas) in the fermentation off-gas.

Example 11 The De-Coupling of Growth and Production of Isoprene in E.coli Expressing Genes from the Mevalonic Acid Pathway and Fermented in aFed-Batch Culture

Example 11 illustrates the de-coupling of cell growth from mevalonicacid and isoprene production.

I. Fermentation Conditions Medium Recipe (Per Liter FermentationMedium):

The medium was generated using the following components per literfermentation medium: K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2 g, citric acidmonohydrate 2 g, ferric ammonium citrate 0.3 g, yeast extract 0.5 g, and1000× modified trace metal solution 1 ml. All of the components wereadded together and dissolved in diH2O. This solution was autoclaved. ThepH was adjusted to 7.0 with ammonium hydroxide (30%) and q.s. to volume.Glucose 10 g, thiamine*HCl 0.1 g, and antibiotics were added aftersterilization and pH adjustment.

1000× Modified Trace Metal Solution:

The 1000× modified trace metal solution was generated using thefollowing components: citric acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g,FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃100 mg, and NaMoO₄*2H₂O 100 mg. Each component was dissolved one at atime in Di H₂O, pH to 3.0 with HCl/NaOH, then q.s. to volume, and filtersterilized with a 0.22 micron filter.

Fermentation was performed with E. coli cells containing thepTrcHis2AUpperPathway (also called pTrcUpperMVA, FIGS. 91 and 92A-92C;SEQ ID NO:23) (50 μg/ml carbenicillin) or the pCL PtrcUpperMVA (alsocalled pCL PtrcUpperPathway (FIG. 26)) (50 μg/ml spectinomycin)plasmids. For experiments in which isoprene was produced, the E. colicells also contained the pTrc KKDyIkIS (50 μg/ml kanamycin) plasmid.These experiments were carried out to monitor mevalonic acid or isopreneformation from glucose at the desired fermentation pH 7.0 andtemperature 30° C. An inoculum of an E. coli strain taken from a frozenvial was streaked onto an LA broth agar plate (with antibiotics) andincubated at 37° C. A single colony was inoculated into tryptone-yeastextract medium. After the inoculum grew to optical density 1.0 whenmeasured at 550 nm, it was used to inoculate the bioreactor.

Glucose was fed at an exponential rate until cells reached thestationary phase. After this time the glucose feed was decreased to meetmetabolic demands. Induction was achieved by adding IPTG. The mevalonicacid concentration in fermentation broth was determined by applyingperchloric acid (Sigma-Aldrich #244252) treated samples (0.3 M incubatedat 4° C. for 5 minutes) to an organic acids HPLC column (BioRad#125-0140). The concentration was determined by comparing the brothmevalonic acid peak size to a calibration curve generated frommevalonolacetone (Sigma-Aldrich #M4667) treated with perchloric acid toform D,L-mevalonate. The isoprene level in the off gas from thebioreactor was determined as described herein. The isoprene titer isdefined as the amount of isoprene produced per liter of fermentationbroth.

II. Mevalonic Acid Production from E. coli BL21 (DE3) Cells Expressingthe pTrcUpperMVA Plasmid at a 150-L Scale

BL21 (DE3) cells that were grown on a plate as explained above inExample 11, part I were inoculated into a flask containing 45 mL oftryptone-yeast extract medium and incubated at 30° C. with shaking at170 rpm for 5 hours. This solution was transferred to a 5-L bioreactorof tryptone-yeast extract medium, and the cells were grown at 30° C. and27.5 rpm until the culture reached an OD₅₅₀ of 1.0. The 5 L of inoculumwas seeded into a 150-L bioreactor containing 45-kg of medium. The IPTGconcentration was brought to 1.1 mM when the OD₅₅₀ reached a value of10. The OD₅₅₀ profile within the bioreactor over time is shown in FIG.60A. The mevalonic acid titer increased over the course of thefermentation to a final value of 61.3 g/L (FIG. 60B). The specificproductivity profile throughout the fermentation is shown in FIG. 60Cand a comparison to FIG. 60A illustrates the de-coupling of growth andmevalonic acid production. The total amount of mevalonic acid producedduring the 52.5 hour fermentation was 4.0 kg from 14.1 kg of utilizedglucose. The molar yield of utilized carbon that went into producingmevalonic acid during fermentation was 34.2%.

III. Mevalonic Acid Production from E. coli BL21 (DE3) Cells Expressingthe pTrcUpperMVA Plasmid at a 15-L Scale

BL21 (DE3) cells that were grown on a plate as explained above inExample 11, part I were inoculated into a flask containing 500 mL oftryptone-yeast extract medium and grown at 30° C. at 160 rpm to OD₅₅₀1.0. This material was seeded into a 15-L bioreactor containing 4.5-kgof medium. The IPTG concentration was brought to 1.0 mM when the OD₅₅₀reached a value of 10. The OD₅₅₀ profile within the bioreactor over timeis shown in FIG. 61A. The mevalonic acid titer increased over the courseof the fermentation to a final value of 53.9 g/L (FIG. 61B). Thespecific productivity profile throughout the fermentation is shown inFIG. 61C and a comparison to FIG. 61A illustrates the de-coupling ofgrowth and mevalonic acid production. The total amount of mevalonic acidproduced during the 46.6 hour fermentation was 491 g from 2.1 kg ofutilized glucose. The molar yield of utilized carbon that went intoproducing mevalonic acid during fermentation was 28.8%.

IV. Mevalonic Acid Production from E. coli FM5 Cells Expressing thepTrcUpperMVA Plasmid at a 15-L Scale

FM5 cells that were grown on a plate as explained above in Example 11,part I were inoculated into a flask containing 500 mL of tryptone-yeastextract medium and grown at 30° C. at 160 rpm to OD₅₅₀ 1.0. Thismaterial was seeded into a 15-L bioreactor containing 4.5-kg of medium.The IPTG concentration was brought to 1.0 mM when the OD₅₅₀ reached avalue of 30. The OD₅₅₀ profile within the bioreactor over time is shownin FIG. 62A. The mevalonic acid titer increased over the course of thefermentation to a final value of 23.7 g/L (FIG. 62B). The specificproductivity profile throughout the fermentation is shown in FIG. 62Cand a comparison to FIG. 62A illustrates the de-coupling of growth andmevalonic acid production. The total amount of mevalonic acid producedduring the 51.2 hour fermentation was 140 g from 1.1 kg of utilizedglucose. The molar yield of utilized carbon that went into producingmevalonic acid during fermentation was 15.2%.

V. Isoprene Production from E. coli BL21 (DE3) Cells Expressing the pCLPtrcUpperMVA and pTrc KKDyIkIS Plasmids at a 15-L Scale

BL21 (DE3) cells expressing the pCL PtrcUpperMVA and pTrc KKDyIkISplasmids that were grown on a plate as explained above in Example 11,part I were inoculated into a flask containing 500 mL of tryptone-yeastextract medium and grown at 30° C. at 160 rpm to OD₅₅₀ 1.0. Thismaterial was seeded into a 15-L bioreactor containing 4.5-kg of medium.The IPTG concentration was brought to 25 μM when the OD₅₅₀ reached avalue of 10. The IPTG concentration was raised to 50 μM when OD₅₅₀reached 190. The IPTG concentration was raised to 100 μM at 38 hours offermentation. The OD₅₅₀ profile within the bioreactor over time is shownin FIG. 63A. The isoprene titer increased over the course of thefermentation to a final value of 2.2 g/L broth (FIG. 63B). The specificproductivity profile throughout the fermentation is shown in FIG. 63Cand a comparison to FIG. 63A illustrates the de-coupling of growth andisoprene production. The total amount of isoprene produced during the54.4 hour fermentation was 15.9 g from 2.3 kg of utilized glucose. Themolar yield of utilized carbon that went into producing isoprene duringfermentation was 1.53%.

VI. Isoprene Production from E. coli BL21 (DE3) Tuner Cells Expressingthe pCL PtrcUpperMVA and pTrc KKDyIkIS Plasmids at a 15-L Scale

BL21 (DE3) tuner cells expressing the pCL PtrcUpperMVA and pTrc KKDyIkISplasmids that were grown on a plate as explained above in Example 11,part I were inoculated into a flask containing 500 mL of tryptone-yeastextract medium and grown at 30° C. at 160 rpm to OD₅₅₀ 1.0. Thismaterial was seeded into a 15-L bioreactor containing 4.5-kg of medium.The IPTG concentration was brought to 26 μM when the OD₅₅₀ reached avalue of 10. The IPTG concentration was raised to 50 μM when OD₅₅₀reached 175. The OD₅₅₀ profile within the bioreactor over time is shownin FIG. 64A. The isoprene titer increased over the course of thefermentation to a final value of 1.3 g/L broth (FIG. 64B). The specificproductivity profile throughout the fermentation is shown in FIG. 64Cand a comparison to FIG. 64A illustrates the de-coupling of growth andisoprene production. The total amount of isoprene produced during the48.6 hour fermentation was 9.9 g from 1.6 kg of utilized glucose. Themolar yield of utilized carbon that went into producing isoprene duringfermentation was 1.34%.

VII. Isoprene Production from E. coli MG1655 Cells Expressing the pCLPtrcUpperMVA and pTrc KKDyIkIS Plasmids at a 15-L Scale

MG1655 cells expressing the pCL PtrcUpperMVA and pTrc KKDyIkIS plasmidsthat were grown on a plate as explained above in Example 11, part I wereinoculated into a flask containing 500 mL of tryptone-yeast extractmedium and grown at 30° C. at 160 rpm to OD₅₅₀ 1.0. This material wasseeded into a 15-L bioreactor containing 4.5-kg of medium. The IPTGconcentration was brought to 24 μM when the OD₅₅₀ reached a value of 45.The OD₅₅₀ profile within the bioreactor over time is shown in FIG. 65A.The isoprene titer increased over the course of the fermentation to afinal value of 393 mg/L broth (FIG. 65B). The specific productivityprofile throughout the fermentation is shown in FIG. 65C and acomparison to FIG. 65A illustrates the de-coupling of growth andisoprene production. The total amount of isoprene produced during the67.4 hour fermentation was 2.2 g from 520 g of utilized glucose. Themolar yield of utilized carbon that went into producing isoprene duringfermentation was 0.92%.

VIII. Isoprene Production from E. coli MG1655ack-pta Cells Expressingthe pCL PtrcUpperMVA and pTrc KKDyIkIS Plasmids at a 15-L Scale

MG1655ack-pta cells expressing the pCL PtrcUpperMVA and pTrc KKDyIkISplasmids that were grown on a plate as explained above in Example 11,part I were inoculated into a flask containing 500 mL of tryptone-yeastextract medium and grown at 30° C. at 160 rpm to OD₅₅₀ 1.0. Thismaterial was seeded into a 15-L bioreactor containing 4.5-kg of medium.The IPTG concentration was brought to 30 μM when the OD₅₅₀ reached avalue of 10. The OD₅₅₀ profile within the bioreactor over time is shownin FIG. 66A. The isoprene titer increased over the course of thefermentation to a final value of 368 mg/L broth (FIG. 66B). The specificproductivity profile throughout the fermentation is shown in FIG. 66Cand a comparison to FIG. 66A illustrates the de-coupling of growth andisoprene production. The total amount of isoprene produced during the56.7 hour fermentation was 1.8 g from 531 g of utilized glucose. Themolar yield of utilized carbon that went into producing isoprene duringfermentation was 0.73%.

IX. Isoprene Production from E. coli FM5 Cells Expressing the pCLPtrcUpperMVA and pTrc KKDyIkIS Plasmids at a 15-L Scale

FM5 cells expressing the pCL PtrcUpperMVA and pTrc KKDyIkIS plasmidsthat were grown on a plate as explained above in Example 11, part I wereinoculated into a flask containing 500 mL of tryptone-yeast extractmedium and grown at 30° C. at 160 rpm to OD₅₅₀ 1.0. This material wasseeded into a 15-L bioreactor containing 4.5-kg of medium. The IPTGconcentration was brought to 27 μM when the OD₅₅₀ reached a value of 15.The OD₅₅₀ profile within the bioreactor over time is shown in FIG. 67A.The isoprene titer increased over the course of the fermentation to afinal value of 235 mg/L broth (FIG. 67B). The specific productivityprofile throughout the fermentation is shown in FIG. 67C and acomparison to FIG. 67A illustrates the de-coupling of growth andisoprene production. The total amount of isoprene produced during the52.3 hour fermentation was 1.4 g from 948 g of utilized glucose. Themolar yield of utilized carbon that went into producing isoprene duringfermentation was 0.32%.

Example 12 Production of Isoprene During the Exponential Growth Phase ofE. coli Expressing Genes from the Mevalonic Acid Pathway and Fermentedin a Fed-Batch Culture

Example 12 illustrates the production of isoprene during the exponentialgrowth phase of cells.

Medium Recipe (Per Liter Fermentation Medium):

The medium was generated using the following components per literfermentation medium: K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2 g, citric acidmonohydrate 2 g, ferric ammonium citrate 0.3 g, yeast extract 0.5 g, and1000× modified trace metal solution 1 ml. All of the components wereadded together and dissolved in diH₂O. This solution was autoclaved. ThepH was adjusted to 7.0 with ammonium hydroxide (30%) and q.s. to volume.Glucose 10 g, thiamine*HCl 0.1 g, and antibiotics were added aftersterilization and pH adjustment.

1000× Modified Trace Metal Solution:

The 1000× modified trace metal solution was generated using thefollowing components: citric acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g,FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃100 mg, and NaMoO₄*2H₂O 100 mg. Each component is dissolved one at atime in Di H2O, pH to 3.0 with HCl/NaOH, then q.s. to volume and filtersterilized with 0.22 micron filter.

Fermentation was performed in a 15-L bioreactor with ATCC 11303 E. colicells containing the pCL PtrcUpperMVA and pTrc KKDyIkIS plasmids. Thisexperiment was carried out to monitor isoprene formation from glucose atthe desired fermentation pH 7.0 and temperature 30° C. An inoculum of E.coli strain taken from a frozen vial was streaked onto an LB broth agarplate (with antibiotics) and incubated at 37° C. A single colony wasinoculated into tryptone-yeast extract medium. After the inoculum grewto OD 1.0, measured at 550 nm, 500 mL was used to inoculate a 5-Lbioreactor.

Glucose was fed at an exponential rate until cells reached thestationary phase. After this time the glucose feed was decreased to meetmetabolic demands. The total amount of glucose delivered to thebioreactor during the 50 hour fermentation was 2.0 kg. Induction wasachieved by adding IPTG. The IPTG concentration was brought to 25 μMwhen the optical density at 550 nm (OD₅₅₀) reached a value of 10. TheIPTG concentration was raised to 50 μM when OD₅₅₀ reached 190. The OD₅₅₀profile within the bioreactor over time is shown in FIG. 99. Theisoprene level in the off gas from the bioreactor was determined asdescribed herein. The isoprene titer increased over the course of thefermentation to a final value of 1.4 g/L (FIG. 100). The total amount ofisoprene produced during the 50 hour fermentation was 10.0 g. Theprofile of the isoprene specific productivity over time within thebioreactor is shown in FIG. 101. The molar yield of utilized carbon thatcontributed to producing isoprene during fermentation was 1.1%. Theweight percent yield of isoprene from glucose was 0.5%.

Example 13 Flammability Modeling and Testing of Isoprene I. Summary ofFlammability Modeling and Testing of Isoprene

Flammability modeling and experiments were performed for varioushydrocarbon/oxygen/nitrogen/water/carbon dioxide mixtures. This modelingand experimental tested was aimed at defining isoprene andoxygen/nitrogen flammability curves under specified steam and carbonmonoxide concentrations at a fixed pressure and temperature. A matrix ofthe model conditions is shown in Table 9, and a matrix of theexperiments performed is shown in Table 5.

TABLE 9 Summary of Modeled Isoprene Flammability Carbon Steam DioxideIsoprene Oxygen Temperature Pressure Concentration ConcentrationConcentration Concentration Series (° C.) (psig) (wt %) (wt. %) (vol. %)(vol. %) A 40 0 0 0 Varying Varying B 40 0 4 0 Varying Varying C 40 0 05 Varying Varying D 40 0 0 10 Varying Varying E 40 0 0 15 VaryingVarying F 40 0 0 20 Varying Varying G 40 0 0 30 Varying Varying

TABLE 10 Summary of Isoprene Flammability Tests Steam Isoprene OxygenTemperature Pressure Concentration Concentration Concentration SeriesNumber (° C.) (psig) (vol. %) (vol. %) (vol. %) 1 40 0 0 Varying Varying2 40 0 4 Varying Varying

II. Description of Calculated Adiabatic Flame Temperature (CAFT) Model

Calculated adiabatic flame temperatures (CAFT) along with a selectedlimit flame temperature for combustion propagation were used todetermine the flammability envelope for isoprene. The computer programused in this study to calculate the flame temperatures is the NASA GlennResearch Center CEA (Chemical Equilibrium with Applications) software.

There are five steps involved in determining the flammability envelopeusing an adiabatic flame temperature model for a homogeneous combustionmechanism (where both the fuel and oxidant are in the gaseous state):selection of the desired reactants, selection of the test condition,selection of the limit flame temperature, modification of the reactants,and construction of a flammability envelope from calculations.

In this first step, selection of desired reactants, a decision must bemade as to the reactant species that will be present in the system andthe quantities of each. In many cases the computer programs used for thecalculations have a list of reactant and product species. If any of thedata for the species to be studied are not found in the program, theymay be obtained from other sources such as the JANAF tables or from theinternet. In this current model data for water, nitrogen, oxygen andcarbon dioxide were present in the program database. The programdatabase did not have isoprene as a species; therefore the thermodynamicproperties were incorporated manually.

The next step is to decide whether the initial pressure and temperatureconditions that the combustion process is taking place in. In this modelthe pressure was 1 atmosphere (absolute) and the temperature was 40° C.,the boiling point of isoprene.

The limit flame temperature for combustion can be either selected basedon theoretical principles or determined experimentally. Each method hasits own limitations.

Based on prior studies, the limit flame temperatures of hydrocarbonsfall in the range of 1000 K to 1500 K. For this model, the value of 1500K was selected. This is the temperature at which the reaction of carbonmonoxide to carbon dioxide (a highly exothermic reaction and constitutesa significant proportion of the flame energy) becomes self sustaining

Once the limit flame temperature has been decided upon, modelcalculations are performed on the given reactant mixture (speciesconcentrations) and the adiabatic flame temperature is determined. Flamepropagation is considered to have occurred only if the temperature isgreater than the limit flame temperature. The reactant mixturecomposition is then modified to create data sets for propagation andnon-propagation mixtures.

This type of model shows good agreement with the experimentallydetermined flammability limits. Regions outside the derived envelope arenonflammable and regions within it are flammable. The shape of theenvelope forms a nose. The nose of the envelope is related to thelimiting oxygen concentration (LOC) for gaseous fuels.

III. Results from Calculated Adiabatic Flame Temperature (CAFT) Model

Plotted in FIGS. 68 through 74 are the CAFT model results for Series Ato G, respectively. The figures plot the calculated adiabatic flametemperature (using the NASA CEA program) as a function of fuelconcentration (by weight) for several oxygen/nitrogen ratios (byweight). The parts of the curve that are above 1500 K, the selectedlimit flame temperature, contain fuel levels sufficient for flamepropagation. The results may be difficult to interpret in the formpresented in FIGS. 68 through 74. Additionally, the current form is notconducive to comparison with experimental data which is generallypresented in terms of volume percent.

Using Series A as an example the data in FIG. 68 can be plotted in theform of a traditional flammability envelope. Using FIG. 68 and readingacross the 1500 K temperature line on the ordinate one can determine thefuel concentration for this limit flame temperature by dropping atangent to the abscissa for each curve (oxygen to nitrogen ratio) thatit intersects. These values can then be tabulated as weight percent offuel for a given weight percent of oxidizer (FIG. 75A). Then knowing thecomposition of the fuel (100 wt. % isoprene) and the composition of theoxidizer (relative content of water, oxygen and nitrogen) molarquantities can be established.

From these molar quantities percentage volume concentrations can becalculated. The concentrations in terms of volume percent can then beplotted to generate a flammability envelope (FIG. 75B). The area boundedby the envelope is the explosible range and the area excluded is thenon-explosible range. The “nose” of the envelope is the limiting oxygenconcentration. FIGS. 76A and 76B contain the calculated volumeconcentrations for the flammability envelope for Series B generated fromdata presented in FIG. 69. A similar approach can be used on datapresented in FIGS. 70-74.

IV. Flammability Testing Experimental Equipment and Procedure

Flammability testing was conducted in a 4 liter high pressure vessel.The vessel was cylindrical in shape with an inner diameter of 6″ and aninternal height of 8.625″. The temperature of the vessel (and the gasesinside) was maintained using external heaters that were controlled by aPID controller. To prevent heat losses, ceramic wool and reflectiveinsulation were wrapped around the pressure vessel. Type K thermocoupleswere used the measure the temperature of the gas space as well as thetemperature of the vessel itself. FIG. 77 illustrates the test vessel.

Before a test was ran, the vessel was evacuated and purged with nitrogento ensure that any gases from previous tests were removed. A vacuum wasthen pulled on the vessel. The pressure after this had been done wastypically around 0.06 bar(a). Due to the nitrogen purging, the gasresponsible for this initial pressure was assumed to be nitrogen. Usingpartial pressures, water, isoprene, nitrogen, and oxygen were then addedin the appropriate amounts to achieve the test conditions in question. Amagnetically driven mixing fan within the vessel ensured mixing of thegaseous contents. The gases were allowed to mix for about 2 minutes withthe fan being turned off approximately 1 minute prior to ignition.

The igniter was comprised of a 1.5 ohm nicrome coil and an AC voltagesource on a timer circuit. Using an oscilloscope, it was determined that34.4 VAC were delivered to the igniter for 3.2 seconds. A maximumcurrent of 3.8 amps occurred approximately halfway into the ignitioncycle. Thus, the maximum power was 131 W and the total energy providedover the ignition cycle was approximately 210 J.

Deflagration data was acquired using a variable reluctance ValidyneDP215 pressure transducer connected to a data acquisition system. A gasmixture was considered to have deflagrated if the pressure rise wasgreater than or equal to 5%.

V. Results of Flammability Testing

The first experimental series (Series 1) was run at 40° C. and 0 psigwith no steam. Running tests at varying concentrations of isoprene andoxygen produced the flammability curve shown in FIG. 78A. The datapoints shown in this curve are only those that border the curve. Adetailed list of all the data points taken for this series is shown inFIGS. 80A and 80B.

FIG. 78B summarizes the explosibility data points shown in FIG. 78A.FIG. 78C is a comparison of the experimental data with the CAFT modelpredicted flammability envelope. The model agrees very well with theexperimental data. Discrepancies may be due to the non-adiabatic natureof the test chamber and limitations of the model. The model looks at aninfinite time horizon for the oxidation reaction and does not take intoconsideration any reaction kinetic limitation.

Additionally, the model is limited by the number of equilibrium chemicalspecies that are in its database and thus may not properly predictpyrolytic species. Also, the flammability envelope developed by themodel uses one value for a limit flame temperature (1500K). The limitflame temperature can be a range of values from 1,000K to 1,500Kdepending on the reacting chemical species. The complex nature ofpyrolytic chemical species formed at fuel concentrations above thestoichiometric fuel/oxidizer level is one reason why the model may notaccurately predict the upper flammable limit for this system.

The second experimental series (Series 2) was run at 40° C. and 0 psigwith a fixed steam concentration of 4%. Running tests at varyingconcentrations of isoprene and oxygen produced the flammability curveshown in FIG. 79A. The data points shown in this curve are only thosethat border the curve. A detailed list of all the data points taken forthis series is shown in FIG. 81. Due to the similarity between the datain Series 1 only the key points of lower flammable limit, limitingoxygen concentration, and upper flammable limits were tested. Theaddition of 4% steam to the test mixture did not significantly changethe key limits of the flammability envelope. It should be noted thathigher concentrations of steam/water and or other inertants mayinfluence the flammability envelope.

FIG. 79B summarizes the explosibility data points shown in FIG. 79A.FIG. 79C is a comparison of the experimental data with the CAFT modelpredicted flammability envelope. The model agrees very well with theexperimental data. Discrepancies may be due to the same factorsdescribed in Series 1

VI. Calculation of Flammability Limits of Isoprene in Air at 3Atmospheres of Pressure

The methods described in Example 13, parts Ito IV were also used tocalculate the flammability limits of isoprene at an absolute systempressure of 3 atmospheres and 40° C. These results were compared tothose of Example 13, parts Ito IV at an absolute system pressure of 1atmosphere and 40° C. This higher pressure was tested because theflammability envelope expands or grows larger as the initial systempressure is increased. The upper flammability limit is affected themost, followed by the limiting oxygen composition. The lowerflammability limit is the least affected (see, for example, “Bulletin627—Flammability Characteristics of Combustible Gases and Vapors”written by Michael G. Zabetakis and published by the former US Bureau ofMines (1965), which is hereby incorporated by reference in its entirety,particular with respect to the calculation of flammability limits).

In FIG. 82, the calculated adiabatic flame temperature is plotted as afunction of isoprene (fuel) concentration, expressed in weight percentof the total fuel/nitrogen/oxygen, where the system pressure wasinitially 3 atmospheres. The calculated flame temperatures are verysimilar to those determined initially in the 1 atmosphere system (FIG.83). As a result, when flammability envelopes are generated using thecalculated adiabatic flammability data, the curves are very similar (seeFIGS. 84 and 85). Therefore, based on these theoretical calculations, asystem pressure increase from 1 atmosphere to 3 atmosphere does notresult in a significant increase/broadening of the flammabilityenvelope. If desired, these model results may be validated usingexperimental testing (such as the experimental testing described hereinat a pressure of 1 atmosphere).

VII. Summary of Flammability Studies

A calculated adiabatic temperature model was developed for theflammability envelope of the isoprene/oxygen/nitrogen/water/carbondioxide system at 40° C. and 0 psig. The CAFT model that was developedagreed well with the experimental data generated by the tests conductedin this work. The experimental results from Series 1 and 2 validated themodel results from Series A and B.

Example 14 Expression Constructs and Strains I. Construction of PlasmidsEncoding Mevalonate Kinase.

A construct encoding the Methanosarcina mazei lower MVA pathway(Accession numbers NC_(—)003901.1, NC_(—)003901.1, NC_(—)003901.1, andNC_(—)003901.1, which are each hereby incorporated by reference in theirentireties) was synthesized with codon optimization for expression in E.coli. This construct is named M. mazei archeal Lower Pathway operon(FIGS. 112A-112C; SEQ ID NO:27) and encodes M. mazei MVK, a putativedecarboxylase, IPK, and IDI enzymes. The gene encoding MVK (Accessionnumber NC_(—)003901.1) was PCR amplified using primers MCM165 and MCM177(Table 11) using the Strategene Herculase II Fusion kit according to themanufacturer's protocol using 30 cycles with an annealing temperature of55° C. and extension time of 60 seconds. This amplicon was purifiedusing a Qiagen PCR column and then digested at 37° C. in a 10 ptreaction with PmeI (in the presence of NEB buffer 4 and BSA). After onehour, NsiI and Roche buffer H were added for an additional hour at 37°C. The digested DNA was purified over a Qiagen PCR column and ligated toa similarly digested and purified plasmid MCM29 (MCM29 is E. coli TOP10(Invitrogen) transformed with pTrcKudzu encoding Kudzu isoprenesynthase) in an 11 μL reaction 5 μL Roche Quick Ligase buffer 1, 1 μLbuffer 2, 1 μL plasmid, 3 μL amplicon, and 1 μL ligase (1 hour at roomtemperature). MCM 29 is pTrcKudzuKan. The ligation reaction wasintroduced into Invitrogen TOP10 cells and transformants selected onLA/kan50 plates incubated at 37° C. overnight. The MVK insert in theresulting plasmid MCM382 was sequenced (FIGS. 113A-113C; SEQ ID NO: 28).

TABLE 11 Oligonucleotides. MCM161 M. mazei MVK forCACCATGGTATCCTGTTCTGCG (SEQ ID NO: 120) MCM162 M. mazei MVK revTTAATCTACTTTCAGACCTTGC (SEQ ID NO: 121) MCM165 M. mazei MVK for w/gcgaacgATGCATaaaggaggtaaaaaaacATGGTATCCTGTTCTG RBSCGCCGGGTAAGATTTACCTG (SEQ ID NO: 122) MCM177 M. mazei MVK rev PstgggcccgtttaaactttaactagactTTAATCTACTTTCAGACCTTGC (SEQ ID NO: 123)

II. Creation of Strains Overexpressing Mevalonate Kinase and IsopreneSynthase.

Plasmid MCM382 was transformed into MCM331 cells (which containschromosomal construct gi1.2KKDyI encoding S. cerevisiae mevalonatekinase, mevalonate phosphate kinase, mevalonate pyrophosphatedecarboxylase, and IPP isomerase) that had been grown to midlog in LBmedium and washed three times in iced, sterile water. One μL of DNA wasadded to 50 μL of cell suspension, and this mixture was electroporatedin a 2 mm cuvette at 2.5 volts, 25 uFd followed immediately by recoveryin 500 μL LB medium for one hour at 37° C. Transformant was selected onLA/kan50 and named MCM391. Plasmid MCM82 was introduced into this strainby the same electroporation protocol followed by selection onLA/kan50/spec50. The resulting strain MCM401 contains a cmp-markedchromosomal construct gi1.2KKDyI, kan-marked plasmid MCM382, andspec-marked plasmid MCM82 (which is pCL PtrcUpperPathway encoding E.faecalis mvaE and mvaS). See Table 12.

TABLE 12 Strains overexpressing mevalonate kinase and isoprene synthase.MCM382 E. coli BL21 (lambdaDE3) pTrcKudzuMVK(M. mazei)GI1.2KKDyI MCM391MCM331 pTrcKudzuMVK(M. mazei) MCM401 MCM331pTrcKudzuMVK(M.mazei)pCLPtrcUpperpathway MCM396 MCM333pTrcKudzuMVK(M. mazei) MCM406MCM333pTrcKudzuMVK(M. mazei)pCLPtrcUpperpathwayIII. Construction of Plasmid MCM376-MVK from M. mazei Archeal Lower inpET200D.

The MVK ORF from the M. mazei archeal Lower Pathway operon (FIGS.112A-112C; SEQ ID NO:27) was PCR amplified using primers MCM161 andMCM162 (Table 11) using the Invitrogen Platinum HiFi PCR mix. 45 μL ofPCR mix was combined with 1 μL template, 1 μL of each primer at 10 μM,and 2 μL water. The reaction was cycled as follows: 94° C. for 2:00; 30cycles of 94° C. for 0:30, 55° C. for 0:30, and 68° C. for 1:15; andthen 72° C. for 7:00, and 4° C. until cool. 3 μL of this PCR reactionwas ligated to Invitrogen pET200D plasmid according to themanufacturer's protocol. 3 μL of this ligation was introduced intoInvitrogen TOP10 cells, and transformants were selected on LA/kan50. Aplasmid from a transformant was isolated and the insert sequenced,resulting in MCM376 (FIGS. 114A-114C; SEQ ID NO:29).

IV. Creation of Expression Strain MCM378.

Plasmid MCM376 was transformed into Invitrogen BL21(DE3) pLysS cellsaccording to the manufacturer's protocol. Transformant MCM378 wasselected on LA/kan50.

Example 15 Production of Isoprene by E. coli Expressing the UpperMevalonic Acid (MVA) Pathway, the Integrated Lower MVA Pathway(gi1.2KKDyI), Mevalonate Kinase from M. mazei, and Isoprene Synthasefrom Kudzu and Grown in Fed-Batch Culture at the 20 mL Batch ScaleMedium Recipe (Per Liter Fermentation Medium):

Each liter of fermentation medium contained K₂HPO₄ 13.6 g, KH₂PO₄ 13.6g, MgSO₄*7H₂O 2 g, citric acid monohydrate 2 g, ferric ammonium citrate0.3 g, (NH₄)₂SO₄ 3.2 g, yeast extract 1 g, and 1000× Trace MetalSolution 1 ml. All of the components were added together and dissolvedin diH₂O. The pH was adjusted to 6.8 with ammonium hydroxide (30%) andbrought to volume. Media was filter sterilized with a 0.22 micronfilter. Glucose (2.5 g) and antibiotics were added after sterilizationand pH adjustment.

1000× Trace Metal Solution:

1000× Trace Metal Solution contained citric Acids*H₂O 40 g, MnSO₄*H₂O 30g, NaC₁₋₁₀ g, FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO₄*7H₂O 1 g, CuSO₄*5H₂O100 mg, H₃BO₃ 100 mg, and NaMoO₄*2H₂O 100 mg. Each component wasdissolved one at a time in di H₂O, pH to 3.0 with HCl/NaOH, then broughtto volume and filter sterilized with a 0.22 micron filter.

Strains:

MCM343 cells are BL21 (DE3) E. coli cells containing the upper mevalonicacid (MVA) pathway (pCL Upper), the integrated lower MVA pathway(gi1.2KKDyI), and isoprene synthase from Kudzu (pTrcKudzu).

MCM401 cells are BL21 (DE3) E. coli cells containing the upper mevalonicacid (MVA) pathway (pCL PtrcUpperPathway), the integrated lower MVApathway (gi1.2KKDyI), and high expression of mevalonate kinase from M.mazei and isoprene synthase from Kudzu (pTrcKudzuMVK(M. mazei)).

Isoprene production was analyzed by growing the strains in 100 mLbioreactors with a 20 mL working volume at a temperature of 30° C. Aninoculum of E. coli strain taken from a frozen vial was streaked onto anLB broth agar plate (with antibiotics) and incubated at 30° C. A singlecolony was inoculated into media and grown overnight. The bacteria werediluted into 20 mL of media to reach an optical density of 0.05 measuredat 550 nm. The 100 mL bioreactors were sealed, and air was pumpedthrough at a rate of 8 mL/min. Adequate agitation of the media wasobtained by stirring at 600 rpm using magnetic stir bars. The off-gasfrom the bioreactors was analyzed using an on-line Hiden HPR-20 massspectrometer. Masses corresponding to isoprene, CO₂, and other gassesnaturally occurring in air were monitored. Accumulated isoprene and CO₂production were calculated by summing the concentration (in percent) ofthe respective gasses over time. Atmospheric CO₂ was subtracted from thetotal in order to estimate the CO₂ released due to metabolic activity.

Isoprene production from a strain expressing the full mevalonic acidpathway and Kudzu isoprene synthase (MCM343) was compared to a strainthat in addition over-expressed MVK from M. mazei and Kudzu isoprenesynthase (MCM401) in 100 mL bioreactors. The bacteria were grown underidentical conditions in defined media with glucose as carbon source.Induction of isoprene production was achieved by addingisopropyl-beta-D-1-thiogalactopyranoside (IPTG) to a final concentrationof either 100 μM or 200 μM. Off-gas measurements revealed that thestrain over-expressing both MVK and isoprene synthase (MCM401) producedsignificantly more isoprene compared to the strain expressing only themevalonic acid pathway and Kudzu isoprene synthase (MCM343) as shown inFIGS. 115A-115D. At 100 μM induction, the MCM401 strain produced 2-foldmore isoprene compared to the MCM343 strain. At 200 μM IPTG induction,the MCM401 strain produced 3.4-fold more isoprene when compared to theMCM343 strain. Analysis of CO₂ in the off-gas from the bioreactors,which is a measure of metabolic activity, indicates that metabolicactivity was independent from IPTG induction and isoprene production.

Example 16 Production of Isoprene by E. coli Expressing the UpperMevalonic Acid (MVA) Pathway, the Integrated Lower MVA Pathway(gi1.2KKDyI), Mevalonate Kinase from M. mazei, and Isoprene Synthasefrom Kudzu and Grown in Fed-Batch Culture at the 15-L Scale MediumRecipe (Per Liter Fermentation Medium):

Each liter of fermentation medium contained K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3 g, yeastextract 0.5 g, and 1000× Modified Trace Metal Solution 1 ml. All of thecomponents were added together and dissolved in DI H₂O. This solutionwas autoclaved. The pH was adjusted to 7.0 with ammonium hydroxide (30%)and q.s. to volume. Glucose 10 g, thiamine*HCl 0.1 g, and antibioticswere added after sterilization and pH adjustment.

1000× Modified Trace Metal Solution:

1000× Modified Trace Metal Solution contained citric Acids*H₂O 40 g,MnSO₄*H₂O 30 g, NaCl 10 g, FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO₄*7H₂O 1g, CuSO₄*5H₂O 100 mg, H₃BO₃ 100 mg, and NaMoO₄*2H₂O 100 mg. Eachcomponent was dissolved one at a time in DI H₂O, pH to 3.0 withHCl/NaOH, then q.s. to volume and filter sterilized with a 0.22 micronfilter.

Fermentation was performed in a 15-L bioreactor with BL21 (DE3) E. colicells containing the upper mevalonic acid (MVA) pathway (pCLPtrcUpperPathway encoding E. faecalis mvaE and mvaS), the integratedlower MVA pathway (gi1.2KKDyI encoding S. cerevisiae mevalonate kinase,mevalonate phosphate kinase, mevalonate pyrophosphate decarboxylase, andIPP isomerase), and high expression of mevalonate kinase from M. mazeiand isoprene synthase from Kudzu (pTrcKudzuMVK(M. mazei)). Thisexperiment was carried out to monitor isoprene formation from glucose atthe desired fermentation pH 7.0 and temperature 30° C. An inoculum of E.coli strain taken from a frozen vial was streaked onto an LB broth agarplate (with antibiotics) and incubated at 37° C. A single colony wasinoculated into tryptone-yeast extract medium. After the inoculum grewto OD 1.0, measured at 550 nm, 500 mL was used to inoculate 5-L ofmedium in a 15-L bioreactor.

Glucose was fed at an exponential rate until cells reached thestationary phase. After this time the glucose feed was decreased to meetmetabolic demands. The total amount of glucose delivered to thebioreactor during the 68 hour fermentation was 3.8 kg. Induction wasachieved by adding isopropyl-beta-D-1-thiogalactopyranoside (IPTG). TheIPTG concentration was brought to 51 μM when the optical density at 550nm (OD₅₅₀) reached a value of 9. The IPTG concentration was raised to 88μM when OD₅₅₀ reached 149. Additional IPTG additions raised theconcentration to 119 μM at OD₅₅₀=195 and 152 μM at OD₅₅₀=210. The OD₅₅₀profile within the bioreactor over time is shown in FIG. 116. Theisoprene level in the off gas from the bioreactor was determined using aHiden mass spectrometer. The isoprene titer increased over the course ofthe fermentation to a final value of 23.8 g/L (FIG. 117). The totalamount of isoprene produced during the 68 hour fermentation was 227.2 gand the time course of production is shown in FIG. 118. The metabolicactivity profile, as measured by TCER, is shown in FIG. 119. The totalviable count (total colony forming units) decreased by two orders ofmagnitude between 10 and 39 hours of fermentation (FIG. 120). The molaryield of utilized carbon that went into producing isoprene duringfermentation was 13.0%. The weight percent yield of isoprene fromglucose was 6.3%.

Example 17 Production of Isoprene by E. coli Expressing the UpperMevalonic Acid (MVA) Pathway, the Integrated Lower MVA Pathway(gi1.2KKDyI), Mevalonate Kinase from M. mazei, and Isoprene Synthasefrom Kudzu and Grown in Fed-Batch Culture at the 15-L Scale (2×100 μMIPTG induction) Medium Recipe (Per Liter Fermentation Medium):

Each liter of fermentation medium contained K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3 g, yeastextract 0.5 g, and 1000× Modified Trace Metal Solution 1 ml. All of thecomponents were added together and dissolved in DI H₂O. This solutionwas autoclaved. The pH was adjusted to 7.0 with ammonium hydroxide (30%)and q.s. to volume. Glucose 10 g, thiamine*HCl 0.1 g, and antibioticswere added after sterilization and pH adjustment.

1000× Modified Trace Metal Solution:

1000× Modified Trace Metal Solution contained citric Acids*H₂O 40 g,MnSO₄*H₂O 30 g, NaCl 10 g, FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO₄*7H₂O 1g, CuSO₄*5H₂O 100 mg, H₃BO₃ 100 mg, and NaMoO₄*2H₂O 100 mg. Eachcomponent was dissolved one at a time in DI H₂O, pH to 3.0 withHCl/NaOH, then q.s. to volume and filter sterilized with a 0.22 micronfilter.

Fermentation was performed in a 15-L bioreactor with BL21 (DE3) E. colicells containing the upper mevalonic acid (MVA) pathway (pCLPtrcUpperPathway encoding E. faecalis mvaE and mvaS), the integratedlower MVA pathway (gi1.2KKDyI encoding S. cerevisiae mevalonate kinase,mevalonate phosphate kinase, mevalonate pyrophosphate decarboxylase, andIPP isomerase), and high expression of mevalonate kinase from M. mazeiand isoprene synthase from Kudzu (pTrcKudzuMVK(M. mazei)). Thisexperiment was carried out to monitor isoprene formation from glucose atthe desired fermentation pH 7.0 and temperature 30° C. An inoculum of E.coli strain taken from a frozen vial was streaked onto an LB broth agarplate (with antibiotics) and incubated at 37° C. A single colony wasinoculated into tryptone-yeast extract medium. After the inoculum grewto OD 1.0, measured at 550 nm, 500 mL was used to inoculate 5-L mediumin a 15-L bioreactor.

Glucose was fed at an exponential rate until cells reached thestationary phase. After this time the glucose feed was decreased to meetmetabolic demands. The total amount of glucose delivered to thebioreactor during the 55 hour fermentation was 1.9 kg. Induction wasachieved by adding isopropyl-beta-D-1-thiogalactopyranoside (IPTG). TheIPTG concentration was brought to 111 μM when the optical density at 550nm (OD₅₅₀) reached a value of 9. The IPTG concentration was raised to193 μM when OD₅₅₀ reached 155. The OD₅₅₀ profile within the bioreactorover time is shown in FIG. 121. The isoprene level in the off gas fromthe bioreactor was determined using a Hiden mass spectrometer. Theisoprene titer increased over the course of the fermentation to a finalvalue of 19.5 g/L (FIG. 122). The total amount of isoprene producedduring the 55 hour fermentation was 133.8 g and the time course ofproduction is shown in FIG. 123. Instantaneous volumetric productivitylevels reached values as high as 1.5 g isoprene/L broth/hr (FIG. 124).Instantaneous yield levels reached as high as 17.7% w/w (FIG. 125). Themetabolic activity profile, as measured by TCER, is shown in FIG. 126.The total viable count (total colony forming units) decreased by twoorders of magnitude between 8 and 36 hours of fermentation (FIG. 127).The molar yield of utilized carbon that went into producing isopreneduring fermentation was 15.8%. The weight percent yield of isoprene fromglucose over the entire fermentation was 7.4%.

In addition, as a control, fermentation was performed in a 15-Lbioreactor with BL21 (DE3) E. coli cells containing the upper mevalonicacid (MVA) pathway (pCL PtrcUpperPathway encoding E. faecalis mvaE andmvaS), the integrated lower MVA pathway (gi1.2KKDyI encoding S.cerevisiae mevalonate kinase, mevalonate phosphate kinase, mevalonatepyrophosphate decarboxylase, and IPP isomerase), and high expression ofmevalonate kinase from M. mazei and isoprene synthase from Kudzu(pTrcKudzuMVK(M. mazei)). This experiment was carried out to monitoruninduced cell metabolic activity as measured by CER from glucose at thedesired fermentation pH 7.0 and temperature 30° C. An inoculum of E.coli strain (MCM401 described above) taken from a frozen vial wasstreaked onto an LB broth agar plate (with antibiotics) and incubated at37° C. A single colony was inoculated into tryptone-yeast extractmedium. After the inoculum grew to OD 1.0, measured at 550 nm, 500 mLwas used to inoculate 5-L medium in a 15-L bioreactor. Glucose was fedat an exponential rate until cells reached the stationary phase. Afterthis time the glucose feed was decreased to meet metabolic demands.

FIG. 148 compares the CER profiles for the uninduced cells describedabove and the cells induced by addingisopropyl-beta-D-1-thiogalactopyranoside (IPTG) in Examples 16 and 17.

Example 18 Production of Isoprene by E. coli Expressing the UpperMevalonic Acid (MVA) Pathway, the Integrated Lower MVA Pathway(gi1.2KKDyI), Mevalonate Kinase from M. mazei, and Isoprene Synthasefrom Kudzu and Grown in Fed-Batch Culture at the 15-L Scale (1×50 μMIPTG+150 μM IPTG Fed Induction) Medium Recipe (Per Liter FermentationMedium):

Each liter of fermentation medium contained K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3 g, yeastextract 0.5 g, and 1000× Modified Trace Metal Solution 1 ml. All of thecomponents were added together and dissolved in diH₂O. This solution wasautoclaved. The pH was adjusted to 7.0 with ammonium hydroxide (30%) andq.s. to volume. Glucose 10 g, thiamine*HCl 0.1 g, and antibiotics wereadded after sterilization and pH adjustment.

1000× Modified Trace Metal Solution:

1000× Modified Trace Metal Solution contained citric Acids*H₂O 40 g,MnSO₄*H₂O 30 g, NaCl 10 g, FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO₄*7H₂O 1g, CuSO₄*5H₂O 100 mg, H₃BO₃ 100 mg, and NaMoO₄*2H₂O 100 mg. Eachcomponent was dissolved one at a time in DI H₂O, pH to 3.0 withHCl/NaOH, then q.s. to volume and filter sterilized with a 0.22 micronfilter.

Fermentation was performed in a 15-L bioreactor with BL21 (DE3) E. colicells containing the upper mevalonic acid (MVA) pathway (pCLPtrcUpperPathway encoding E. faecalis mvaE and mvaS), the integratedlower MVA pathway (gi1.2KKDyI encoding S. cerevisiae mevalonate kinase,mevalonate phosphate kinase, mevalonate pyrophosphate decarboxylase, andIPP isomerase), and high expression of mevalonate kinase from M. mazeiand isoprene synthase from Kudzu (pTrcKudzuMVK(M. mazei)). Thisexperiment was carried out to monitor isoprene formation from glucose atthe desired fermentation pH 7.0 and temperature 30° C. An inoculum of E.coli strain taken from a frozen vial was streaked onto an LB broth agarplate (with antibiotics) and incubated at 37° C. A single colony wasinoculated into tryptone-yeast extract medium. After the inoculum grewto OD 1.0, measured at 550 nm, 500 mL was used to inoculate 5-L mediumin a 15-L bioreactor.

Glucose was fed at an exponential rate until cells reached thestationary phase. After this time the glucose feed was decreased to meetmetabolic demands. The total amount of glucose delivered to thebioreactor during the 55 hour fermentation was 2.2 kg. Induction wasachieved by adding isopropyl-beta-D-1-thiogalactopyranoside (IPTG). TheIPTG concentration was brought to 51 μM when the optical density at 550nm (OD₅₅₀) reached a value of 10. In addition to the IPTG spike, atOD₅₅₀=10 a constant feed began and delivered 164 mg of IPTG over 18hours. The OD₅₅₀ profile within the bioreactor over time is shown inFIG. 128. The isoprene level in the off gas from the bioreactor wasdetermined using a Hiden mass spectrometer. The isoprene titer increasedover the course of the fermentation to a final value of 22.0 g/L (FIG.129). The total amount of isoprene produced during the 55 hourfermentation was 170.5 g and the time course of production is shown inFIG. 130. The metabolic activity profile, as measured by TCER, is shownin FIG. 131. When the airflow to the bioreactor was decreased from 8slpm to 4 slpm for a period of about 1.7 hours, the concentration ofisoprene in the offgas increased from 0.51 to 0.92 w/w % (FIG. 132).These elevated levels of isoprene did not appear to have any negativeimpact on cell metabolic activity as measured by the total carbondioxide evolution rate (TCER), since TCER declined only 7% between 37.2and 39.3 hours (FIG. 132). The total viable count (total colony formingunits) decreased by two orders of magnitude between 7 and 36 hours offermentation (FIG. 133). The molar yield of utilized carbon that wentinto producing isoprene during fermentation was 16.6%. The weightpercent yield of isoprene from glucose over the entire fermentation was7.7%.

Example 19 The Effect of Externally Applied Isoprene on a Wild-Type E.coli Grown in Fed-Batch Culture at the 1-L Scale Medium Recipe (PerLiter Fermentation Medium):

Each liter of fermentation medium contained K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3 g, yeastextract 0.5 g, and 1000× Modified Trace Metal Solution 1 ml. All of thecomponents were added together and dissolved in diH₂O. This solution wasautoclaved. The pH was adjusted to 7.0 with ammonium hydroxide (30%) andq.s. to volume. Glucose 10 g, thiamine*HCl 0.1 g, and antibiotics wereadded after sterilization and pH adjustment.

1000× Modified Trace Metal Solution:

1000× Modified Trace Metal Solution contained citric Acids*H₂O 40 g,MnSO₄*H₂O 30 g, NaCl 10 g, FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO₄*7H₂O 1g, CuSO₄*5H₂O 100 mg, H₃BO₃ 100 mg, and NaMoO₄*2H₂O 100 mg. Eachcomponent was dissolved one at a time in DI H₂O, pH to 3.0 withHCl/NaOH, then q.s. to volume and filter sterilized with a 0.22 micronfilter.

Fermentation was performed in a 1-L bioreactor with BL21 (DE3) E. colicells. This experiment was carried out to monitor the effects ofisoprene on cell viability and metabolic activity in a glucose fed-batchbioreactor at the desired fermentation pH 7.0 and temperature 30° C. Aninoculum of E. coli strain from a frozen vial was inoculated intotryptone-yeast extract medium. After the inoculum grew to OD 1.0,measured at 550 nm, 50 mL was used to inoculate 0.5-L medium in a 1-Lbioreactor.

Glucose was fed at an exponential rate until cells reached thestationary phase. After this time the glucose feed was fed to meetmetabolic demands. Isoprene was fed into the bioreactor using nitrogengas as a carrier. The rate of isoprene feeding was 1 g/L/hr duringmid-growth phase (OD₅₅₀=31-44) and lasted for a total of 75 minutes(13.2 to 14.4 hours). The OD₅₅₀ profile within the bioreactor over timeis shown in FIG. 134. The metabolic activity profile, as measured byTCER, is shown in FIG. 135. The total viable count (total colony formingunits) increased by 14-fold during the period when isoprene wasintroduced into the bioreactor (FIG. 136).

Example 20 Production of Isoprene and Expression of Isoprene Synthase bySaccharomyces cerevisiae

The Kudzu isoprene synthase enzyme was optimized for expressionaccording to a hybrid Saccharomyces cerevisiae/Pichia pastoris codonusage table, synthesized, and cloned into pDONR221:19430 (by DNA 2.0,FIG. 140 for map and FIG. 141 for sequence (SEQ ID NO:38)). A Gateway®Cloning (Invitrogen) reaction was performed according to themanufacturer's protocol: Since pDONR221:19430 was an “entry” vector, theLR Clonase II enzyme (the LR Reaction) was used to introduce thecodon-optimized isoprene synthase into the “destination” vectorpYES-DEST52 (Invitrogen).

The LR Reaction was then transformed into Top10 chemically competentcells (Invitrogen) according to the manufacturer's protocol, andbacteria harboring pYES-DEST52 plasmids with the isoprene synthase ORFwere selected for on LA plates containing 50 μg/ml carbenicillin.Individual positive transformants were tested by colony PCR (see belowfor primer concentrations and thermocycling parameters) using illustraPuReTaq Ready-To-Go™ PCR Beads (GE Healthcare) with the T7 forwardprimer and the Yeast isoprene synthase-Rev2 primer (See Table 13).

TABLE 13 Primer sequences for amplifying isoprene synthase. Primer NameSequence (5′ to 3′) Purpose Yeast HGS - CACCAAAGACTTCATAGACTForward primer for yeast For2 (SEQ ID NO: 124)optimized isoprene synthase Yeast HGS - AGAGATATCTTCCTGCTGCTReverse primer for yeast Rev2 (SEQ ID NO: 125)optimized isoprene synthase T7 Forward TAATACGACTCACTATAGGGPCR and sequencing primer (SEQ ID NO: 126)

Plasmids that yielded a PCR fragment of the correct size (1354 bp) werepurified by miniprep (Qiagen) and sent for sequencing (QuintaraBiosciences, Berkeley, Calif.) with the T7 Forward and Yeast isoprenesynthase-For2 primers (See Table 13). Results from sequencing runs werecompared to the known sequence of pDONR221:19430 (using Vector NTIsoftware, Invitrogen), and a single plasmid, pDW14, was selected forfurther study (FIG. 142A for map and FIGS. 142B and C for the completesequence (SEQ ID NO:39)). The sequence of pDW14 diverged from that ofpDONR221:19430 by a single nucleotide (marked in bold in FIG. 142B). Thesingle nucleotide change (G to A) did not result in a change in the ORF,since it was in the third position of a lysine-encoding codon.

Purified pDW14 was transformed into Saccharomyces cerevisiae strainINVSc-1 using the protocol described in the S. c. EasyCompTransformation kit (Invitrogen). INVSc-1 strains harboring pDW14 orpYES-DEST52 (which contains an intact URA3 gene) were selected for andmaintained on SC Minimal Medium with 2% glucose without uracil, asdescribed in the pYES-DEST52 Gateway Vector manual (Invitrogen). Twoindependent isolates of INVSc-1 containing pDW14 and a single controlstrain with pYES-DEST52 were chosen for further analysis.

To induce isoprene synthase expression, cultures were grown overnight inliquid SC Minimal Medium. The cultures were then diluted to an OD₆₀₀ ofapproximately 0.2 and grown for 2-3 hours. Cultures were spun bycentrifugation, washed once, resuspended in an equal volume (10 ml) ofSC minimal medium with 1% raffinose, 2% galactose without uracil, andgrown overnight to induce the expression of isoprene synthase. The OD₆₀₀of the strains was determined (FIG. 144A), and strains were harvested bycentrifugation and resuspended in 2 ml of lysis buffer (a 1:1 mix of 50%glycerol and PEB pH 7.4: Tris Base 2.423 g/L, MgCl₂ (Anhydrous) 1.904g/L, KCl 14.910 g/L, DTT 0.154 g/L, Glycerol 50 mL/L).

The lysis mixtures were passed through a french press three times, andlysates were analyzed by SDS-PAGE. For Coomassie gel analysis (FIG.143A), samples were diluted 1:1 with 2×SDS loading buffer with reducingagent, loaded (20 μl total volume) onto a 4-12% bis-tris gel, run in MESbuffer, and stained using SimplyBlue SafeStain according to themanufacturer's protocol (the Invitrogen Novex system).

The WesternBreeze kit (Invitrogen) was used for transfer and chromogenicdetection of isoprene synthase on a nitrocellulose membrane. The primaryantibody was 1799A 10 week diluted 1:1000 in Invitrogen antibodydiluent. Primary antibody binding was followed by development with asecondary antibody labeled with Alexa Fluor 488 (Invitrogen Catalog No.A-11008) to permit quantitative signal determination. The western blotprocedure was carried out as described by Invitrogen. The fluorescencesignal was recorded with a Molecular Dynamics Storm instrument using theblue filter setting and quantitatively analyzed with the MolecularDynamics ImageQuant image analysis software package. Specific activityof the library members was calculated from the ratio of the amount ofisoprene produced divided by either the A600 of the induction culturesor the isoprene synthase protein concentration determined by westernblot. FIG. 143B shows that isoprene synthase was present in the inducedINVSc-1 strains harboring pDW14 (lanes 2 and 3) in comparison to thecontrol harboring pYES-DEST52 (lane 1).

The DMAPP assay for isoprene synthase headspace was performed on 25 μLof the lysate from each strain for which 5 μL 1 M MgCl2, 5 μL 100 mMDMAPP, and 65 μL 50 mM Tris pH 8 were added. The reaction was performedat 30° C. for 15 minutes in a gas tight 1.8 mL GC tube. Reactions wereterminated by addition of 100 μL 250 mM EDTA pH 8. FIG. 144B showed thespecific activity values (in μg HG/L/OD) of the induced strainsharboring pDW14 in comparison to the control. Induced strains harboringpDW14 displayed approximately 20× higher activity than the controllacking isoprene synthase.

PCR Cycling Parameters

Illustra PuReTaq Ready-To-Go™ PCR Beads (GE Healthcare) were used witholigonucleotide primer pairs at a concentration of 0.4 μM each in 25 μltotal volume/reaction. For analysis of plasmids resulting from the LRClonase reaction (Invitrogen), a small amount of bacteria fromindividual colonies on a selective plate was added to each tubecontaining the PCR mix described above. The reaction cycle was asfollows: 1) 95° C. for 4 minutes; 2) 95° C. for 20 seconds; 3) 52° C.for 20 seconds; 4) 72° C. for 30 seconds; 5 cycles of steps 2 through 4;5) 95° C. for 20 seconds; 6) 55° C. for 20 seconds; 7) 72° C. for 30seconds; 25 cycles of steps 5 through 7, 72° C. for 10 minutes, and 4°C. until cool.

Example 21 Production of Isoprene in Pseudomonas and Other Gram NegativeBacteria Construction of pBBRSHGSOpt2_(—)2, conjugation in Pseudomonasand Measurement of Isoprene Synthase Activity

A gene encoding isoprene synthase from Pueraria lobata (Kudzu plant) wascodon-optimized for different microbial species of interest (Table 14;fluo-opt2v2 was the sequence chosen) and was synthesized by DNA2.0,Menlo Park, Calif. The map and sequence of fluo-opt2v2 can be found inFIGS. 145A and 145B (SEQ ID NO:40). HindIII and BamHI restriction siteswere added to the synthesized sequence for easier cloning, and a RBS wasadded in front of the ATG to enhance transcription.

Number of rare codons, as a function of the microbial species, indifferent versions of codon-optimized isoprene synthase from Puerarialobata. Several rounds of optimization led to a gene with no rare codonsin the all the species of interest.

TABLE 14 Number of rare codons. fluo-opt1 Organism (quote) fluo-opt2fluo-opt3 E. coli opt fluo-opt2v2 Pseudomonas fluorescens Pf-5 19 X X 570 Phodopseudomonas palustris 37 13 3 74 0 CGA009 Pseudomonas putida F1 0 0 0 29 0 Corynebacterium glutamicum 4 (Ser) 0 0 0 0 (ATCC)Pseudomonas fluorescens PfO-1 1 (Val) 0 0 57 0

The gene was provided by DNA2.0 in a cloning vector. The vector wasdigested with HindIII/BamHI, the band corresponding to the insert ofinterest was gel-purified, and relegated with HindIII/BamHI-digestedpBBR1MCS5 (Kovach et al, Gene 166:175-176, 1995, which is incorporatedby reference in its entirety, particularly with respect to pBBR1MCS5),FIG. 146A for map and FIGS. 146B and C for sequence (SEQ ID NO:41). Thisresulted in plasmid pBBR5HGSOpt2_(—)2 (FIG. 147A for map and FIGS. 147Band C for sequence (SEQ ID NO:42)) in which isoprene synthase wasexpressed from the lac promoter presented in pBBR1MCS5.

The vector was transformed in E. coli 517-1 and mated with Pseudomonasputida F1 ATCC700007 and Pseudomonas fluorescens ATCC 13525. Afterconjugation on LB, selection for plasmid-harboring Pseudomonas strainswas on M9+16 mM sodium citrate+Gentamicin 50 μg/ml. Presence of theplasmid in the strains thus generated was checked by plasmid preparationusing the Qiagen kit (Valencia, Calif.).

Isoprene synthase activities of the recombinant strains P. putida,pBBR5HGSOpt2_(—)2 and P. fluorescens, pBBR5HGSOpt2_(—)2 were assayed bygrowing the strains in TM3 medium (as described in Example 1 Part II)+10g/L glucose, harvesting the biomass in mid-log phase, breaking the cellsby French Press and proceeding with the DMAPP assay. Results of theassay were presented in Table 15. The presence of activity measured bythe DMAPP assay confirmed that isoprene synthase was expressed inPseudomonas.

Isoprene synthase activity was examined in Pseudomonas putida andPseudomonas fluorescens expressing isoprene synthase from the lacpromoter, using plasmid pBBR5HGSOpt2_(—)2

TABLE 15 Isoprene synthase activity in Pseudomonas putida andPseudomonas fluorescens. Isoprene synthase activity Strain OD mgisoprene/(L · h · OD) P. fluorescens, pBBR5HGSOpt2_2 1.46 0.96 P.putida, pBBR5HGSOpt2_2 3.44 0.65 Control (P. putida w/o plasmid) 8.32 Tobe determined

Example 22 Growth of E. coli and Pseudomonas Strains on Sugar CaneCompared to Glucose, and Expression of Isoprene Synthase Using BothSubstrates I. Preparation of Liquid Sugar Cane

Crystallized raw cane sugar was dissolved in water in the following way:750 g H₂O was added to 250 g sugar. The solution was stirred and gentlyheated until dissolution. Some material was not soluble. The weight ofthe solution was adjusted to 1 kg after dissolution to replenish theevaporated water. The volume of the solution was measured to be 940 mL.Hence the concentration of the solution was 265 g/L. The product labelclaimed 14 g of carbohydrate for 15 g of raw sugar cane. Hence thecarbohydrate concentration of the solution was 248 g/L. Dry solids weremeasured to be 24.03%, close enough of the expected 250 g/kg. pH of thesolution was 5.49. Glucose concentration was measured using anenzymatic/spectrophotometric assay, with glucose oxidase. The glucoseconcentration was 17.4 g/L.

As a majority of microorganisms do not use sucrose, but can use glucoseand fructose, the solution was split in two. One half was autoclavedonce for 30 minutes (sugar cane as is). Some inversion resulted, as theglucose content increased to 29.75 g/L (See FIG. 149). The other half ofthe solution was adjusted to pH 4.0 using phosphoric acid, then thesolution was inverted by autoclaving (inverted sugar cane). Three cyclesof 30 min were sufficient to obtain complete inversion, as shown on FIG.149. Both solutions were used for the growth curves described below.

II. Growth Curves of Different Strains of E. coli and Pseudomonas onSugar Cane Compared to Glucose

One colony of each of the strains presented in Table 16 was inoculatedin 25 ml TM3+10 g/L glucose, and was grown overnight at 30° C. and 200rpm. TM3 is described in Example 7, Section II. The morning after, 1 mlof each culture was used to inoculate flasks containing 25 mL TM3 and 10g/L glucose, 10 g/L sugar cane as is, or 10 g/L inverted sugar cane(sugar cane solutions described above). The flasks were incubated at 30°C. and 200 rpm and samples were taken regularly to measure OD600. FIGS.150 and 151 show that growth rate and biomass yield were comparable forglucose and inverted sugar cane, both for Pseudomonas and E. colistrains. P. fluorescens showed some signs of being able to use sugarcane which has not been inverted too.

TABLE 16 Strains used in this study. Strain Escherichia coli BL21 MG1655ATCC11303 B REL 606 Pseudomonas putida F1 (ATCC700007) Fluorescens(ATCC13525)III. Comparison of Isoprene Production from E. coli Expressing IsopreneSynthase when Grown on Glucose or Sugar Cane

E. coli MCM401 (BL21(DE3)) containing the full MVA pathway, mevalonatekinase from M. mazei and isoprene synthase from Pueraria lobata, asdescribed in Example 14, Section II was grown in TM3+either 10 g/Lglucose or 10 g/L inverted sugar cane (based on carbohydrateconcentration of the syrup). Flasks were inoculated from an overnightculture on TM3+10 g/L glucose at an OD₆₀₀=0.2. Antibiotics were addedwhere needed. After two hours, the E. coli cultures were induced with400 μM IPTG. After 6 hours of growth, isoprene production and isoprenesynthase activities, using the DMAPP assay as described in Example 2B,were measured. Results are presented in Table 17 and illustrate clearlythat inverted sugar cane is equivalent to glucose in terms of isopreneand isoprene synthase production on a per cell basis.

TABLE 17 Isoprene Isoprene synthase activity production mg mg isoprene/Strain Carbon Source OD isoprene/(L · h · OD) (L · h · OD) MCM401Glucose 2.20 21.06 8.98 MCM401 Sugar cane 2.32 20.20 9.23 inverted

Example 23 Construction of E. coli Strains Expressing the S. cerevisiaegi1.2KKDyI Operon, P. alba Isoprene Synthase, M. mazei MevalonateKinase, pCL Upper MVA (E. faecalis mvaE and mvaS) and ybhE (pgl) (i)Construction of Strain EWL201 (BL21, Cm-GI1.2-KKDyI)

E. coli BL21 (Novagen brand, EMD Biosciences, Inc.) was a recipientstrain, transduced with MCM331 P1 lysate (lysate prepared according tothe method described in Ausubel, et al., Current Protocols in MolecularBiology. John Wiley and Sons, Inc.). MCM331 cells contain chromosomalconstruct gi1.2KKDyI encoding S. cerevisiae mevalonate kinase,mevalonate phosphate kinase, mevalonate pyrophosphate decarboxylase, andIPP isomerase (i.e., the gi1.2-KKDyI operon from S. cerevisiae).Transductants were selected for by spreading cells onto L Agar and 20μg/μl chloramphenicol. The plates were incubated overnight at 30° C.Analysis of transductants showed no colonies on control plates (water+cells control plate for reversion and water and P1 lysate control platefor lysate contamination.

Four transductants were picked and used to inoculate 5 mL L Broth and 20μg/μl chloramphenicol. The cultures were grown overnight at 30° C. withshaking at 200 rpm. To make genomic DNA preps of each transductant forPCR analysis, 1.5 mL of overnight cell culture were centrifuged. Thecell pellet was resuspended with 400 μl Resuspension Buffer (20 mM Tris,1 mM EDTA, 50 mM NaCl, pH 7.5) and 4 μl RNase, DNase-free (Roche) wasadded. The tubes were incubated at 37° C. for 30 minutes followed by theaddition of 4 μl 10% SDS and 4 μl of 10 mg/ml Proteinase K stocksolution (Sigma-Aldrich). The tubes were incubated at 37° C. for 1 hour.The cell lysate was transferred into 2 ml Phase Lock Light Gel tubes(Eppendorf) and 200 μl each of saturated phenol pH7.9 (Ambion Inc.) andchloroform were added. The tubes were mixed well and microcentrifugedfor 5 minutes. A second extraction was done with 400 μl chloroform andthe aqueous layer was transferred to a new eppendorf tube. The genomicDNA was precipitated by the addition of 1 ml of 100% ethanol andcentrifugation for 5 minutes. The genomic DNA pellet was washed with 1ml 70% ethanol. The ethanol was removed and the genomic DNA pellet wasallowed to air dry briefly. The genomic DNA pellet was resuspended with200 μl TE.

Using Pfu Ultra II DNA polymerase (Stratagene) and 200 ng/μl of genomicDNA as template, 2 different sets of PCR reaction tubes were preparedaccording to manufacturer's protocol. For set 1, primers MCM130 and GBCm-Rev (Table 18) were used to ensure transductants were successfullyintegrated into the attTn7 locus. PCR parameters for set 1 were 95° C.for 2 minutes (first cycle only), 95° C. for 25 seconds, 55° C. for 25seconds, 72° C. for 25 seconds (repeat steps 2-4 for 28 cycles), 72° C.for 1 minute. For set 2, primers MVD For and MVD Rev (Table 18) wereused to ensure that the gi1.2-KKDyI operon integrated properly. PCRparameters for set 2 were 95° C. for 2 minutes (first cycle only), 95°C. for 25 seconds, 55° C. for 25 seconds, 72° C. for 10 seconds (repeatsteps 2-4 for 28 cycles), 72° C. for 1 minute. Analysis of PCR ampliconson a 1.2% E-gel (Invitrogen Corp.) showed that all 4 transductant cloneswere correct. One was picked and designated as strain EWL201.

(ii) Construction of Strain EWL204 (BL21, loopout-GI1.2-KKDyI)

The chloramphenicol marker was looped out of strain EWL201 using plasmidpCP20 as described by Datsenko and Wanner (2000) (Datsenko et al., ProcNatl. Acad. Sci. USA 97:6640-6645, 2000). One-step inactivation ofchromosomal genes in Escherichia coli K-12 using PCR products. (Datsenkoet al., PNAS, 97: 6640-6645, 2000). EWL201 cells were grown in L Brothto midlog phase and then washed three times in ice-cold, sterile water.An aliquot of 50 μl of cell suspension was mixed with 1 μl of pCP20 andthe cell suspension mixture was electroporated in a 2 mm cuvette(Invitrogen Corp.) at 2.5 Volts and 25 uFd using a Gene PulserElectroporator (Bio-Rad Inc.). 1 ml of LB was immediately added to thecells, then transferred to a 14 ml polypropylene tube (Sarstedt) with ametal cap. Cells were allowed to recover by growing for 1 hour at 30° C.Transformants were selected on L Agar and 20 μg/μl chloramphenicol and50 μg/μl carbenicillin and incubated at 30° C. overnight. The next day,a single clone was grown in 10 ml L Broth and 50 μg/μl carbenicillin at30° C. until early log phase. The temperature of the growing culture wasthen shifted to 42° C. for 2 hours. Serial dilutions were made, thecells were then spread onto LA plates (no antibiotic selection), andincubated overnight at 30° C. The next day, 20 colonies were picked andpatched onto L Agar (no antibiotics) and LA and 20 μg/μl chloramphenicolplates. Plates were then incubated overnight at 30° C. Cells able togrow on LA plates, but not LA and 20 μg/μl chloramphenicol plates, weredeemed to have the chloramphenicol marker looped out (picked one anddesignated as strain EWL204).

(iii) Construction of Plasmid pEWL230 (pTrc P. alba)

Generation of a synthetic gene encoding Populus alba isoprene synthase(P. alba HGS) was outsourced to DNA2.0 Inc. (Menlo Park, Calif.) basedon their codon optimization method for E. coli expression. The syntheticgene was custom cloned into plasmid pET24a (Novagen brand, EMDBiosciences, Inc.) and delivered lyophilized (FIGS. 152, 153A-B; SEQ IDNO:43).

A PCR reaction was performed to amplify the P. alba isoprene synthase(P. alba HGS) gene using pET24 P. alba HGS as the template, primersMCM182 and MCM192, and Herculase II Fusion DNA polymerase (Stratagene)according to manufacturer's protocol. PCR conditions were as follows:95° C. for 2 minutes (first cycle only), 95° C. for 25 seconds, 55° C.for 20 seconds, 72° C. for 1 minute, repeat for 25 cycles, with finalextension at 72° C. for 3 minutes. The P. alba isoprene synthase PCRproduct was purified using QIAquick PCR Purification Kit (Qiagen Inc.).

P. alba isoprene synthase PCR product was then digested in a 20 μlreaction containing 1 μl BspHI endonuclease (New England Biolabs) with 2μl 10×NEB Buffer 4. The reaction was incubated for 2 hours at 37° C. Thedigested PCR fragment was then purified using the QIAquick PCRPurification Kit. A secondary restriction digest was performed in a 20μl reaction containing 1 μl PstI endonuclease (Roche) with 4,110× BufferH. The reaction was incubated for 2 hours at 37° C. The digested PCRfragment was then purified using the QIAquick PCR Purification Kit.Plasmid pTrcHis2B (Invitrogen Corp.) was digested in a 20 μl reactioncontaining 1 μl NcoI endonuclease (Roche), 1 μl PstI endonuclease, and 2μl 10× Buffer H. The reaction was incubated for 2 hours at 37° C. Thedigested pTrcHis2B vector was gel purified using a 1.2% E-gel(Invitrogen Corp.) and extracted using the QIAquick Gel Extraction Kit(Qiagen) (FIG. 154). Using the compatible cohesive ends of BspHI andNcoI sites, a 20 μl ligation reaction was prepared containing 5 μl P.alba isoprene synthase insert, 2 μl pTrc vector, 1 μl T4 DNA ligase (NewEngland Biolabs), 2 μl 10× ligase buffer, and 10 μl ddH₂O. The ligationmixture was incubated at room temperature for 40 minutes. The ligationmixture was desalted by floating a 0.025 μm nitrocellulose membranefilter (Millipore) in a petri dish of ddH₂O and applying the ligationmixture gently on top of the nitrocellulose membrane filter for 30minutes at room temperature. MCM446 cells (see Section II) were grown inLB to midlog phase and then washed three times in ice-cold, sterilewater. An aliquot of 50 μl of cell suspension was mixed with 5 μl ofdesalted pTrc P. alba HGS ligation mix. The cell suspension mixture waselectroporated in a 2 mm cuvette at 2.5 Volts and 25 uFd using a GenePulser Electroporator. 1 ml of LB is immediately added to the cells,then transferred to a 14 ml polypropylene tube (Sarstedt) with a metalcap. Cells were allowed to recover by growing for 2 hour at 30° C.Transformants were selected on L Agar and 50 μg/μl carbenicillin and 10mM mevalonic acid and incubated at 30° C. The next day, 6 transformantswere picked and grown in 5 ml L Broth and 50 μg/μl carbenicillin tubesovernight at 30° C. Plasmid preps were performed on the overnightcultures using QIAquick Spin Miniprep Kit (Qiagen). Due to the use ofBL21 cells for propagating plasmids, a modification of washing the spincolumns with PB Buffer 5× and PE Buffer 3× was incorporated to thestandard manufacturer's protocol for achieving high quality plasmid DNA.Plasmids were digested with PstI in a 20 μl reaction to ensure thecorrect sized linear fragment. All 6 plasmids were the correct size andshipped to Quintara Biosciences (Berkeley, Calif.) for sequencing withprimers MCM65, MCM66, EL1000 (Table 18). DNA sequencing results showedall 6 plasmids were correct. One plasmid was picked designated asplasmid EWL230 (FIGS. 155, 156A-B; SEQ ID NO:44).

iv) Construction of Plasmid pEWL244 (pTrc P. alba-mMVK)

A PCR reaction was performed to amplify the Methanosarcina mazei (M.mazei) MVK gene using MCM376 as the template (see section (v) below),primers MCM165 and MCM177 (see Table 18), and Pfu Ultra II Fusion DNApolymerase (Stratagene) according to manufacturer's protocol. PCRconditions were as follows: 95° C. for 2 minutes (first cycle only), 95°C. for 25 seconds, 55° C. for 25 seconds, 72° C. for 18 seconds, repeatfor 28 cycles, with final extension at 72° C. for 1 minute. The M. mazeiMVK PCR product was purified using QIAquick PCR Purification Kit (QiagenInc.).

The M. mazei MVK PCR product was then digested in a 40 μl reactioncontaining 8 μl PCR product, 2 μl PmeI endonuclease (New EnglandBiolabs), 4 μl 10×NEB Buffer 4, 4 μl 10×NEB BSA, and 22 μl of ddH₂O. Thereaction was incubated for 3 hours at 37° C. The digested PCR fragmentwas then purified using the QIAquick PCR Purification Kit. A secondaryrestriction digest was performed in a 47 μl reaction containing 2 μlNsiI endonuclease (Roche), 4.7 μl 10× Buffer H, and 40 μl of PmeIdigested M. mazei MVK fragment. The reaction was incubated for 3 hoursat 37° C. The digested PCR fragment was then gel purified using a 1.2%E-gel and extracted using the QIAquick Gel Extraction Kit. PlasmidEWL230 was digested in a 40 μl reaction containing 10 μl plasmid, 2 μlPmeI endonuclease, 4 μl 10×NEB Buffer 4, 4 μl 10×NEB BSA, and 20 μl ofddH₂O. The reaction was incubated for 3 hours at 37° C. The digested PCRfragment was then purified using the QIAquick PCR Purification Kit. Asecondary restriction digest was performed in a 47 μl reactioncontaining 2 μl PstI endonuclease, 4.7 μl 10× Buffer H, and 40 μl ofPmeI digested EWL230 linear fragment. The reaction was incubated for 3hours at 37° C. The digested PCR fragment was then gel purified using a1.2% E-gel and extracted using the QIAquick Gel Extraction Kit (FIG.157). Using the compatible cohesive ends of NsiI and PstI sites, a 20 μlligation reaction was prepared containing 8 μl M. mazei MVK insert, 3 μlEWL230 plasmid, 1 μl T4 DNA ligase, 2 μl 10× ligase buffer, and 6 μlddH₂O. The ligation mixture was incubated overnight at 16° C. The nextday, the ligation mixture was desalted by floating a 0.025 μmnitrocellulose membrane filter in a petri dish of ddH₂O and applying theligation mixture gently on top of the nitrocellulose membrane filter for30 minutes at room temperature. MCM446 cells were grown in LB to midlogphase and then washed three times in ice-cold, sterile water. An aliquotof 50 μl of cell suspension was mixed with 5 μl of desalted pTrc P.alba-mMVK ligation mix. The cell suspension mixture was electroporatedin a 2 mm cuvette at 2.5 Volts and 25 uFd using a Gene PulserElectroporator. 1 ml of LB is immediately added to the cells, then thecells are transferred to a 14 ml polypropylene tube with a metal cap.Cells were allowed to recover by growing for 2 hour at 30° C.Transformants were selected on LA and 50 μg/μl carbenicillin and 5 mMmevalonic acid plates and incubated at 30° C. The next day, 6transformants were picked and grown in 5 ml LB and 50 μg/μlcarbenicillin tubes overnight at 30° C. Plasmid preps were performed onthe overnight cultures using QIAquick Spin Miniprep Kit. Due to the useof BL21 cells for propagating plasmids, a modification of washing thespin columns with PB Buffer 5× and PE Buffer 3× was incorporated to thestandard manufacturer's protocol for achieving high quality plasmid DNA.Plasmids were digested with PstI in a 20 μl reaction to ensure thecorrect sized linear fragment. Three of the 6 plasmids were the correctsize and shipped to Quintara Biosciences for sequencing with primersMCM65, MCM66, EL1000, EL1003, and EL1006 (Table 18). DNA sequencingresults showed all 3 plasmids were correct. One was picked anddesignated as plasmid EWL244 (FIGS. 158 and 159A-B; SEQ ID NO:45).

v) Construction of Plasmid MCM376-MVK from M. mazei Archaeal Lower inpET200D.

The MVK ORF from the M. mazei archaeal Lower Pathway operon (FIGS.160A-C; SEQ ID NO:46) was PCR amplified using primers MCM161 and MCM162(Table 18) using the Invitrogen Platinum HiFi PCR mix. 45 μL of PCR mixwas combined with 1 μL template, 1 μL of each primer at 10 μM, and 2 μLwater. The reaction was cycled as follows: 94° C. for 2:00 minutes; 30cycles of 94° C. for 0:30 minutes, 55° C. for 0:30 minutes and 68° C.for 1:15 minutes; and then 72° C. for 7:00 minutes, and 4° C. untilcool. 3 μL of this PCR reaction was ligated to Invitrogen pET200Dplasmid according to the manufacturer's protocol. 3 μL of this ligationwas introduced into Invitrogen TOP10 cells, and transformants wereselected on LA/kan50. A plasmid from a transformant was isolated and theinsert sequenced, resulting in MCM376 (FIGS. 161A-C).

vi) Construction of Strain EWL251 (BL21(DE3), Cm-GI1.2-KKDyI, pTrc P.alba-mMVK)

MCM331 cells (which contain chromosomal construct gi1.2KKDyI encoding S.cerevisiae mevalonate kinase, mevalonate phosphate kinase, mevalonatepyrophosphate decarboxylase, and IPP isomerase) were grown in LB tomidlog phase and then washed three times in ice-cold, sterile water.Mixed 50 μl of cell suspension with 1 μl of plasmid EWL244. The cellsuspension mixture was electroporated in a 2 mm cuvette at 2.5 Volts and25 uFd using a Gene Pulser Electroporator. 1 ml of LB is immediatelyadded to the cells, and then the cells were transferred to a 14 mlpolypropylene tube with a metal cap. Cells were allowed to recover bygrowing for 2 hours at 30° C. Transformants were selected on LA and 50μg/μl carbenicillin and 5 mM mevalonic acid plates and incubated at 37°C. One colony was selected and designated as strain EWL251.

vii) Construction of Strain EWL256 (BL21(DE3), Cm-GI1.2-KKDyI, pTrc P.alba-mMVK, pCL Upper MVA)

EWL251 cells were grown in LB to midlog phase and then washed threetimes in ice-cold, sterile water. Mixed 50 μl of cell suspension with 1μl of plasmid MCM82 (comprising pCL PtrcUpperPathway (also known as “pCLUpper MVA”), encoding E. faecalis mvaE and mvaS). Plasmid pCL Ptrc UpperPathway was constructed as described in Example 8 above. The cellsuspension mixture was electroporated in a 2 mm cuvette at 2.5 Volts and25 gd using a Gene Pulser Electroporator. 1 ml of LB was immediatelyadded to the cells. Cells were then transferred to a 14 ml polypropylenetube with a metal cap. Cells were allowed to recover by growing for 2hours at 30° C. Transformants were selected on LA and 50 μg/μlcarbenicillin and 50 μg/μl spectinomycin plates and incubated at 37° C.One colony was picked and designated as strain EWL256.

TABLE 18 Primer Sequences Primer name Primer sequence MCM130ACCAATTGCACCCGGCAGA (SEQ ID NO: 127) GB CmGCTAAAGCGCATGCTCCAGAC (SEQ ID NO: 128) Rev MVDGACTGGCCTCAGATGAAAGC (SEQ ID NO: 129) For MVDCAAACATGTGGCATGGAAAG (SEQ ID NO: 130) Rev MCM182GGGCCCGTTTAAACTTTAACTAGACTCTGCAGTTAGCGTTCAAACGGCAGAA (SEQ ID NO: 131)MCM192 CGCATGCATGTCATGAGATGTAGCGTGTCCACCGAAAA (SEQ ID NO: 132) MCM65ACAATTTCACACAGGAAACAGC (SEQ ID NO: 133) MCM66CCAGGCAAATTCTGTTTTATCAG (SEQ ID NO: 106) EL1000GCACTGTCTTTCCGTCTGCTGC (SEQ ID NO: 134) MCM165GCGAACGATGCATAAAGGAGGTAAAAAAACATGGTATCCTGTTCTGCGCCGGGTAAGATTTACCTG (SEQ ID NO: 122) MCM177GGGCCCGTTTAAACTTTAACTAGACTTTAATCTACTTTCAGACCTTGC (SEQ ID NO: 123) EL1003GATAGTAACGGCTGCGCTGCTACC (SEQ ID NO: 137) EL1006GACAGCTTATCATCGACTGCACG (SEQ ID NO: 138) MCM161CACCATGGTATCCTGTTCTGCG (SEQ ID NO: 120) MCM162TTAATCTACTTTCAGACCTTGC (SEQ ID NO: 121)viii) Construction of Strain RM111608-2 (Cm-GI1.2-KKDyI, pTrc P.alba-mMVK, pCL Upper MVA, pBBRCMPGI1.5-pgl)

The BL21 strain of E. coli producing isoprene (EWL256) was constructedwith constitutive expression of the ybhE gene (encoding E. coli6-phosphogluconolactonase) on a replicating plasmidpBBR1MCS5(Gentamycin) (obtained from Dr. K. Peterson, Louisiana StateUniversity).

FRT-based recombination cassettes, and plasmids for Red/ET-mediatedintegration and antibiotic marker loopout were obtained from GeneBridges GmbH (Germany). Procedures using these materials were carriedout according to Gene Bridges protocols. Primers Pgl-F (SEQ ID NO:139)and PglGI1.5-R (SEQ ID NO:140) were used to amplify the resistancecassette from the FRT-gb2-Cm-FRT template using Stratagene Herculase IIFusion kit according to the manufacturer's protocol. The PCR reaction(50 μl, final volume) contained: 5 μL buffer, 1 μL template DNA(FRT-gb2-Cm-F from Gene Bridges), 10 pmols of each primer, and 1.5 μL 25mM dNTP mix, made to 50 μL with dH₂O. The reaction was cycled asfollows: 1×2 minutes, 95° C. then 30 cycles of (30 seconds at 95° C.; 30seconds at 63° C.; 3 minutes at 72° C.).

The resulting PCR product was purified using the QiaQick PCRpurification kit (Qiagen) and electroporated into electrocompetentMG1655 cells harboring the pRed-ET recombinase-containing plasmid asfollows. Cells were prepared by growing in 5 mLs of L broth to andOD600-0.6 at 30° C. The cells were induced for recombinase expression bythe addition of 4% arabinose and allowed to grow for 30 minutes at 30°C. followed by 30 minutes of growth at 37° C. An aliquot of 1.5 mLs ofthe cells was washed 3-4 times in ice cold dH₂O. The final cell pelletwas resuspended in 40 μL of ice cold dH₂O and 2-5 μL of the PCR productwas added. The electroporation was carried out in 1-mm gap cuvettes, at1.3 kV in a Gene Pulser Electroporator (Bio-Rad Inc.). Cells wererecovered for 1-2 hours at 30° C. and plated on L agar containingchloramphenicol (5 μg/mL). Five transformants were analyzed by PCR andsequencing using primers flanking the integration site (2 primer sets:pgl and 49 rev and 3′ EcoRV-pglstop; Bottom Pgb2 and Top GB's CMP(946)). A correct transformant was selected and this strain wasdesignated MG1655 GI1.5-pgl::CMP.

The chromosomal DNA of MG1655 GI1.5-pgl::CMP was used as template togenerate a PCR fragment containing the FRT-CMP-FRT-GI1.5-ybhE construct.This construct was cloned into pBBR1MCS5(Gentamycin) as follows. Thefragment, here on referred to as CMP-GI1.5-pgl, was amplified using the5′ primer Pglconfirm-F (SEQ ID NO:141) and 3′ primer 3′ EcoRV-pglstop(SEQ ID NO:142). The resulting fragment was cloned using the InvitrogenTOPO-Blunt cloning kit into the plasmid vector pCR-Blunt II-TOPO assuggested from the manufacturer. The NsiI fragment harboring theCMP-GI1.5-pgl fragment was cloned into the PstI site of pBBR1MCS5(Gentamycin). A 20 μl ligation reaction was prepared containing 5 μlCMP-GI1.5-pgl insert, 2 μl pBBR1MCS5 (Gentamycin) vector, 1 μl T4 DNAligase (New England Biolabs), 2 μl 10× ligase buffer, and 10 μl ddH₂O.The ligation mixture was incubated at room temperature for 40 minutesthen 2-4 μL were electroporated into electrocompetent Top10 cells(Invitrogen) using the parameters disclosed above. Transformants wereselected on L agar containing 10 μg/ml chloramphenicol and 5 μg/mlGentamycin. The sequence of the selected clone was determined using anumber of the primers described above as well as with the in-house T3and Reverse primers provided by Sequetech, CA. This plasmid wasdesignated pBBRCMPGI1.5-pgl (FIGS. 162, 163A-B and SEQ ID NO:48).

Plasmid pBBRCMPGI1.5-pgl was electroporated into EWL256, as describedherein and transformants were plated on L agar containingChloramphenicol (10 μg/mL), Gentamycin (5 lag/mL), spectinomycin (50μg/mL), and carbenicillin (50 μg/mL). One transformant was selected anddesignated strain RM111608-2.

Primers:

Pgl-F (SEQ ID NO: 139)5′-ACCGCCAAAAGCGACTAATTTTAGCTGTTACAGTCAGTTGAATTAACCCTCACTAAAGGGCGGCCGC-3′ PglGI1.5-R (SEQ ID NO: 140)5′-GCTGGCGATATAAACTGTTTGCTTCATGAATGCTCCTTTGGGTTACCTCCGGGAAACGCGGTTGATTTGTTTAGTGGTTGAATTATTTGCTCAGGATGTGGCATAGTCAAGGGCGTGACGGCTCGCTAATACGACTCACTATAGGG CTCGAG-3′ 3′EcoRV-pglstop: (SEQ ID NO: 142)5′-CTT GAT ATC TTA GTG TGC GTT AAC CAC CAC (SEQ ID NO: 136)pgl +49 rev: CGTGAATTTGCTGGCTCTCAG (SEQ ID NO: 135)Bottom Pgb2: GGTTTAGTTCCTCACCTTGTC (SEQ ID NO: 92)Top GB's CMP (946): ACTGAAACGTTTTCATCGCTC Pglconfirm-F (SEQ ID NO: 141)5′-ACCGCCAAAAGCGACTAATTTTAGCT-3′

Example 24 Improvement of Isoprene Production by Constitutive Expressionof ybhE (pgl) in E. coli

This example shows production of isoprene in a strain constitutivelyexpressing E. coli ybhE (pgl) compared to a control strain expressingybhE at wild-type levels (i.e., EWL256). The gene ybhE (pgl) encodes E.coli 6-phosphogluconolactonase that suppresses posttranslationalgluconylation of heterologously expressed proteins and improves productsolubility and yield while also improving biomass yield and flux throughthe pentose phosphate pathway (Aon et al., Applied and EnvironmentalMicrobiology, 74(4): 950-958, 2008).

i) Small Scale Analysis

Media Recipe (per liter fermentation media): K₂HPO₄ 13.6 g, KH₂PO₄ 13.6g, MgSO₄*7H₂O 2 g, citric acid monohydrate 2 g, ferric ammonium citrate0.3 g, (NH₄)₂SO₄ 3.2 g, yeast extract 1 g, 1000× Trace Metals Solution 1ml. All of the components were added together and dissolved in diH₂O.The pH was adjusted to 6.8 with ammonium hydroxide (30%) and brought tovolume. Media was filter-sterilized with a 0.22 micron filter. Glucose5.0 g and antibiotics were added after sterilization and pH adjustment.

1000× Trace Metal Solution (per liter fermentation media): CitricAcid*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g, FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1g, ZnSO₄.7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃ 100 mg, NaMoO₄*2H₂O 100 mg.Each component is dissolved one at a time in diH₂O. The pH is adjustedto 3.0 with HCl/NaOH, and then the solution is brought to volume andfilter-sterilized with a 0.22 micron filter.

(a) Experimental Procedure

Isoprene production was analyzed by growing the strains in a Cellerator™from MicroReactor Technologies, Inc. The working volume in each of the24 wells was 4.5 mL. The temperature was maintained at 30° C., the pHsetpoint was 7.0, the oxygen flow setpoint was 20 sccm and the agitationrate was 800 rpm. An inoculum of E. coli strain taken from a frozen vialwas streaked onto an LB broth agar plate (with antibiotics) andincubated at 30° C. A single colony was inoculated into media withantibiotics and grown overnight. The bacteria were diluted into 4.5 mLof media with antibiotics to reach an optical density of 0.05 measuredat 550 nm.

Off-gas analysis of isoprene was performed using a gaschromatograph-mass spectrometer (GC-MS) (Agilent) headspace assay.Sample preparation was as follows: 100 μL of whole broth was placed in asealed GC vial and incubated at 30° C. for a fixed time of 30 minutes.Following a heat kill step, consisting of incubation at 70° C. for 5minutes, the sample was loaded on the GC.

Optical density (OD) at a wavelength of 550 nm was obtained using amicroplate reader (Spectramax) during the course of the run. Specificproductivity was obtained by dividing the isoprene concentration (μg/L)by the OD reading and the time (hour).

The two strains EWL256 and RM11608-2 were assessed at 200 and 400 μMIPTG induction levels. Samples were analyzed for isoprene production andcell growth (OD₅₅₀) at 1, 2.5, 4.75, and 8 hours post-induction. Sampleswere done in duplicate.

(b) Results

The experiment demonstrated that at 2 different concentrations of IPTGthe strain expressing the ybhE (pgl) had a dramatic 2-3 fold increase inspecific productivity of isoprene compared to the control strain.

ii) Isoprene Fermentation from E. coli Expressing Cm-GI1.2-KKDyI, M.mazei Mevalonate Kinase, P. alba Isoprene Synthase, and ybhE (pgl)(RM111608-2) and Grown in Fed-Batch Culture at the 15-L Scale

Medium Recipe (per liter fermentation medium): K₂HPO₄ 7.5 g, MgSO₄*7H₂O2 g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3 g, yeastextract 0.5 g, 1000× Modified Trace Metal Solution 1 ml. All of thecomponents were added together and dissolved in diH₂O. This solution wasautoclaved. The pH was adjusted to 7.0 with ammonium hydroxide (30%) andq.s. to volume. Glucose 10 g, thiamine*HCl 0.1 g, and antibiotics wereadded after sterilization and pH adjustment.

1000× Modified trace Metal Solution: Citric Acids*H₂O 40 g, MnSO₄*H₂O 30g, NaCl 10 g, FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO₄*7H₂O 1 g, CuSO₄*5H₂O100 mg, H₃BO₃ 100 mg, NaMoO₄*2H₂O 100 mg. Each component is dissolvedone at a time in Di H₂O, pH to 3.0 with HCl/NaOH, then q.s. to volumeand filter sterilized with a 0.22 micron filter

Fermentation was performed in a 15-L bioreactor with BL21 (DE3) E. colicells containing the upper mevalonic acid (MVA) pathway (pCL Upper), theintegrated lower MVA pathway (gi1.2KKDyI), high expression of mevalonatekinase from M. mazei and isoprene synthase from P. alba (pTrcAlba-mMVK),and high expression of E. coli pgl (pBBR-pgl). This experiment wascarried out to monitor isoprene formation from glucose at the desiredfermentation pH 7.0 and temperature 34° C. A frozen vial of the E. colistrain was thawed and inoculated into tryptone-yeast extract medium.After the inoculum grew to OD 1.0, measured at 550 nm, 500 mL was usedto inoculate a 15-L bioreactor bringing the initial volume to 5-L.

Glucose was fed at an exponential rate until cells reached thestationary phase. After this time the glucose feed was decreased to meetmetabolic demands. The total amount of glucose delivered to thebioreactor during the 40 hour (59 hour) fermentation was 3.1 kg (4.2 kgat 59 hour). Induction was achieved by adding IPTG. The IPTGconcentration was brought to 110 μM when the optical density at 550 nm(OD₅₅₀) reached a value of 4. The IPTG concentration was raised to 192μM when OD₅₅₀ reached 150. The OD₅₅₀ profile within the bioreactor overtime is shown in FIG. 164A. The isoprene level in the off gas from thebioreactor was determined using a Hiden mass spectrometer. The isoprenetiter increased over the course of the fermentation to a maximum valueof 33.2 g/L at 40 hours (48.6 g/L at 59 hours) (FIG. 164B). The isoprenetiter increased over the course of the fermentation to a maximum valueof 40.0 g/L at 40 hours (60.5 g/L at 59 hours) (FIG. 164C). The totalamount of isoprene produced during the 40-hour (59-hour) fermentationwas 281.3 g (451.0 g at 59 hours) and the time course of production isshown in FIG. 164D. The time course of volumetric productivity is shownin FIG. 164E and shows that an average rate of 1.0 g/L/hr was maintainedbetween 0 and 40 hours (1.4 g/L/hour between 19 and 59 hour). Themetabolic activity profile, as measured by CER, is shown in FIG. 164F.The molar yield of utilized carbon that went into producing isopreneduring fermentation was 19.6% at 40 hours (23.6% at 59 hours). Theweight percent yield of isoprene from glucose was 8.9% at 40 hours(10.7% at 59 hours).

Example 25 Co-Production of Isoprene and Hydrogen in E. coli StrainsExpressing M. mazei Mevalonate Kinase, P. alba Isoprene Synthase, pCLUpper MVA (E. faecalis mvaE and mvaS) and vbhE (pgl) Collection andAnalysis of Fermentation Off-Gas for Hydrogen and Isoprene Levels

Fermentations were performed using strains RM111608-2 (E. coli BL21(DE3), pCL

Upper MVA, cmR-gi1.2-yKKDyI, pTrcAlba-mMVK, pBBR cmR-gi1.5-pgl) and EWL256 (E. coli BL21 (DE3), pCL Upper MVA, cmR-gi1.2-yKKDyI,pTrcAlba-mMVK). Construction of bacterial strains is described inExample 23 above.

Large scale production of isoprene from E. coli was determined from afed-batch culture of E. coli strains EWL256 and RM111608-2 expressing M.mazei mevalonate kinase, P. alba isoprene synthase, pCL Upper MVA (E.faecalis mvaE and mvaS) and either constitutively expressing ybhE (pgl)(RM111608-2) or normally expressing ybhE (pgl) (EWL256). This experimentdemonstrates that growing cells in the presence of glucose resulted inthe co-production of isoprene and hydrogen.

The recipe for the fermentation medium (TM2) per liter of TM2fermentation medium was as follows: K₂HPO₄ 13.6 g, KH₂PO₄ 13.6 g,MgSO₄*7H₂O 2 g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3g, (NH₄)₂SO₄ 3.2 g, yeast extract 5 g, 1000× Modified Trace MetalSolution 1 ml. 1000× Modified Trace Metal Solution: Citric Acids*H₂O 40g, MnSO₄*H₂O 30 g, NaCl 10 g, FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO₄*7H₂O1 g, CuSO₄*5H₂O 100 mg, H₃BO₃ 100 mg, NaMoO₄*2H₂O 100 mg. For the 1000×Modified Trace Metal Solution, each component is dissolved one at a timein Di H₂O, pH to 3.0 with HCl/NaOH, then brought to final volume indistilled water and filter sterilized with a 0.22 micron (μm) filter(this solution is not autoclaved). For the TM2 fermentation medium, allof the components were added together, dissolved in diH₂O, the pH wasadjusted to 6.8 with potassium hydroxide (KOH), q.s. to volume, and themedium was filter sterilized with a 0.22 micron (μm) filter. Glucose wassourced from Cargill as 99DE (dextrose equivalent), 71% DS (dry solids)syrup.

Fermentations were performed in 15-L bioreactors with E. coli strainsEWL256 or RM111608-2, containing the upper mevalonic acid (MVA) pathway(pCL Upper MVA), the integrated lower MVA pathway (cmR-gi1.2-yKKDyI),mevalonate kinase from M. mazei and isoprene synthase from P. alba(pTrcAlba-mMVK), and constitutively expressing ybhE (pgl) (RM111608-2)or normally expressing ybhE (pgl) (EWL256). This experiment was carriedout to monitor isoprene formation from glucose at the desiredfermentation conditions (pH 7.0 and temperature 34° C.).

An inoculum of the appropriate E. coli strain taken from a frozen vialwas prepared in peptone-yeast extract medium. After the inoculum grew toOD₅₅₀=0.6, 600 mL was used to inoculate a 15-L bioreactor containing TM2medium. Glucose was fed at an exponential rate until cells reached thestationary phase. After this time the glucose feed was decreased to meetmetabolic demands. The total amount of glucose delivered to thebioreactor during the 67 hour fermentation was 3.9 kg. Induction wasachieved by adding isopropyl-beta-D-1-thiogalactopyranoside (IPTG). TheIPTG concentration was brought to 102 μM when the optical density at 550nm (OD₅₅₀) reached a value of 9. The IPTG concentration was raised to192 μM when OD₅₅₀ reached 140. At various times after inoculation,samples were removed and the amount of isoprene produced was determinedas described below. Levels of hydrogen, nitrogen, oxygen, carbondioxide, and isoprene in the off gas from the bioreactor were determinedusing a Hiden HPR-20 mass spectrometer as discussed below.

Samples of fermentation off-gas from 15-L bioreactors were collectedinto 20 mL glass headspace vials by sparging the vials at 1L_(offgas)/min for 10 seconds and sealed with metal screw caps fittedwith teflon-coated septa (Agilent, CA). The vials were analyzed within30 minutes of collection.

Analysis of the two samples was performed by infusion into a HidenHPR-20 mass spectrometer (Hiden Analytics, U.K.) at a rate of 4 scc/min(4 mL/min) by placing the inlet tube of the mass spectrometer into theuncapped headspace vials for 1-2 minutes. The HPR-20 instrument wasconfigured to scan masses corresponding to hydrogen (m/z 2), nitrogen(m/z 28), oxygen (m/z 32), carbon dioxide (m/z 44) and isoprene (m/z67). The Faraday detector was used for masses 28, 32, 44 and 67. The SEMdetector was used for hydrogen (m/z 2). Detector response was measuredin arbitrary units of pressure (Torr). Absolute hydrogen levels wereestimated by comparison to an authentic hydrogen gas standard. Resultswere recorded using MASsoft V 6.21.0.51 software (Hiden Analytics,United Kingdom).

Results

Off-gas samples were taken from two fermentation runs and analyzed asdescribed above:

A) Strain RM111608-2 (E. coli BL21 (DE3), pCL upper, cmR-gi1.2-yKKDyI,pTrcAlba-mMVK, pBBR cmR-gi1.5-pgl). Sample was taken at 64.8 hours intothe run during which time the fermentation was being run anaerobicallywith a nitrogen sparge at 1 vvm.

B) Strain EWL256 (E. coli BL21 (DE3), pCL upper, cmR-gi1.2-yKKDyI,pTrcAlba-mMVK). Sample was taken at 34.5 hours into the run during whichtime the fermentation was being run aerobically with an air sparge at 1vvm.

The results are depicted in FIGS. 165A-B. In both cases low levels ofhydrogen were detected, in addition to isoprene, oxygen and carbondioxide. The baseline reading for hydrogen was 0.95×10⁻⁸ Ton. BothSample A and B gave reading of around 1.3×10⁻⁸ Ton. Based on acomparison to a hydrogen standard, the amount of hydrogen present in theoff-gas for samples A and B was estimated to be less than 10 ppmv (partsper million volume) but above the baseline. As shown in FIGS. 165A-B,both samples A and B also contained significant amounts of isoprene andcarbon dioxide.

Example 26 Co-Production of Isoprene and Hydrogen in E. coli StrainsExpressing M. mazei Mevalonate Kinase, P. alba Isoprene Synthase, pCLUpper MVA (E. faecalis mvaE and mvaS) and vbhE (pgl) Collection andAnalysis of Fermentation Off-Gas for Hydrogen and Isoprene Levels

The objective of this experiment is co-produce hydrogen and isoprene inan engineered strain of E. coli. For this purpose, a portion of the hycoperon encoding E. coli hydrogenase-3 will be expressed in strain EWL256[BL21 (DE3), pCL upper, cmR-gi1.2-yKKDyI, pTrcAlba-mMVK], prepared asdescribed herein, although any of the bacterial strains describedherein, such as RM111608-2, can be similarly modified. An expressionconstruct comprising hyc operon genes hycB (gi|16130631), hycC(gi|16130630), hycD (gi|16130629), hycE (gi|16130628), hycF(gi|16130627), and hycG (gi|16130626) is prepared by standard cloningmethods known in the art based upon publicly available gene sequences,and introduced into strain EWL256 to produce new strain EWL256+Hyd-3.

The impact of additional mutations on co-production of hydrogen andisoprene is assessed alone or in combination in EWL256+Hyd-3, byintroducing genes involved in the maturation or regulation ofhydrogenase-3 (e.g., hycH (gi|16130625) and hycl (gi|16130624)), byinactivating or deleting genes involved in hydrogen uptake or transport(e.g., E. coli hydrogenase-1 (hya operon) and hydrogenase-2 (hyboperon)) or related proteins (e.g., formate dehydrogenase (fdhF(gi|16130624)), repressor of formate lyase (hycA (gi|16130632)), formatedehydrogenase N, alpha subunit (fdnG (gi|16129433)), formatedehydrogenase 0, large subunit (fdoG (gi|16131734)), nitrate reductase(narG (gi|16129187)), fumarate reductase regulator (fnr (gi|16129295)),and acetyl-coenzyme A synthetase (acs (gi|16131895))), by activatinggenes involved in upregulation of hydrogenases (e.g., activator offormate hydrogen lyase (fhlA (gi|16130638)), by inactivating or deletinggenes involved in the production of fermentation side products (e.g.,lactate dehydrogenase (ldhA (gi|16129341)), fumarate reductase membraneprotein (frdC (gi|16131977)), alcohol dehydrogenase (adhE(gi|16129202)), pyruvate oxidase (poxB (gi|16128839)), pyruvatedehydrogenase El component ackA/pta (aceE (gi|16128107)), formatedehydrogenase regulatory protein (hycA (gi|16130632)), and formatetransporters A and B (FocA (gi|16128871) and FocB (gi|16130417)), or byexpression of heterologous genes involved in hydrogen metabolism (e.g.,glyceraldehyde-3-phosphate dehydrogenase from Clostridium acetobutylicum(gapC (gi|15893997)).

Fermentations are performed using engineered variants of strain EWL256+Hyd-3 (BL21 (DE3), pCL upper, cmR-gi1.2-yKKDyI, pTrcAlba-mMVK andhycB-F), modified to comprise one or more additional mutations asdescribed herein, either alone or in combination, essentially asdescribed in Example 25 above. Co-production of hydrogen and isoprene isassessed by analysis of off-gas samples essentially as described above.Strains are selected for further analysis based upon the rate ofisoprene and hydrogen co-production.

Unless defined otherwise, the meanings of all technical and scientificterms used herein are those commonly understood by one of skill in theart to which this invention belongs. Singleton, et al., Dictionary ofMicrobiology and Molecular Biology, 2nd ed., John Wiley and Sons, NewYork (1994), and Hale & Marham, The Harper Collins Dictionary ofBiology, Harper Perennial, N.Y. (1991) provide one of skill with ageneral dictionary of many of the terms used in this invention. It is tobe understood that this invention is not limited to the particularmethodology, protocols, and reagents described, as these may vary. Oneof skill in the art will also appreciate that any methods and materialssimilar or equivalent to those described herein can also be used topractice or test the invention.

Example 27 Production of Isoprene or Mevalonate from Fatty Acid or PalmOil in E. coli fadR atoC LS5218 Containing the Upper or Upper and LowerMevalonic Acid Pathway Plus Kudzu Isoprene Synthase

Escherichia coli fadR atoC strain LS5218 (#6966) was obtained from theColi Genetic Stock Center. FadR encodes a transcription repressor thatnegatively regulates expression of the genes encoding fatty aciddegradation enzymes (Campbell et al., J. Bacteriol. 183: 5982-5990,2001). AtoC is a response regulator in a two-component regulatory systemwherein AtoS regulates acetolactate metabolism. The fadR atoC strainallows constitutive expression of the fatty acid degradation genes andincorporates long chain fatty acids into long-chain-lengthpolyhydroxyalkanoates. When palm oil is used as a carbon source foreither mevalonate or isoprene production, the palm oil was converted toglycerol plus fatty acid. Methods for this are well known in the art,and it can be done either enzymatically by incubation with a lipase (forexample Porcine pancreatic lipase, Candida rugosa lipase, or othersimilar lipases) or chemically by saponification with a base such assodium hydroxide.

i) E. coli fadR atoC Strain Expressing the Upper Mevalonic Acid Pathway

Strain WW4 was created by electroporating pCLPtrcUpperPathway intoLS5218 using standard methods (Sambrooke et al., Molecular Cloning: ALaboratory Manual, 2^(nd) ed., Cold Spring Harbor, 1989). Incorporationof the plasmid was demonstrated by the production of mevalonic acid(MVA) when cells were cultured in TM3 medium supplemented with eitherC12 fatty acid (FA) or palm oil as the carbon source. To demonstrateproduction of MVA by WW4 from fatty acid, cells from an overnightculture were diluted 1 to 100 into 5 mL of modified TM3 medium (TM3without yeast extract) supplemented with 0.25% C12 FA (Sigma cat#L9755). The first sign of MVA production (24 mg/L) was apparent afterovernight incubation at 30° C. of the IPTG induced culture. Productionincreased over three days with the final level of 194 mg/L of MVAproduced. To demonstrate production of MVA by WW4 from oil, cells froman overnight culture were diluted 1 to 100 into modified TM3 mediumsupplemented with 200 mg of digested palm oil per 5 mL of TM3 medium.The first sign of MVA production (50 mg/L) was apparent after overnightincubation of the IPTG induced culture at 30° C. Production increasedover three days with a final level of 500 mg/L of MVA produced.

ii) E. coli fadR atoC Strain Expressing the Upper and Lower MVA PathwayPlus Kudzu Isoprene Synthase

Escherichia coli strain WW4 (LS5218 fadR atoC pCLPtrcUpperPathway) wastransformed with pMCM118 [pTrcKKDyIkIS] to yield WW10. The incorporationof the plasmid was demonstrated by evidence of production of isoprenewhen the strain was cultured in TM3 and glucose and induced with IPTG(100, 300, or 900 μM). The strain was relatively sensitive to IPTG andshowed a significant growth defect even at 100 μM IPTG. These resultsare shown in FIG. 70A.

To test isoprene production from dodecanoic acid, WW10 was culturedovernight in L broth containing spectinomycin (50 μg/ml), and kanamycin(50 μg/ml) at 37 C with shaking at 200 rpm. The cells were washed withmodified TM3 medium by centrifugation and resuspension in their originalculture volume with this medium. The washed and resuspended cells fromthis starter culture were diluted 1 to 100 and 1 to 10 into 5 mL ofmodified TM3 medium containing 0.125% C12 Fatty Acid (Sigma cat #L9755).

To demonstrate production of mevalonate from palm oil, the oil waspredigested with lipase at 37° C. and 250 rpm for several days torelease the fatty acids (evidence of hydrolysis was judged by the foamformed when tubes were shaken).

In addition, a culture was set up by diluting the washed cells at 1 to10 into modified TM3 medium contained in test tubes with palm oil. Afurther tube was set up by the addition of 0.125% C12FA to the remainder(2.5 mL) of the washed cells without further dilution (bioconversion).After 3.75 hours of growth at 30° C. with shaking at 250 rpm all of thecultures were induced by the addition of 50 μM IPTG. Incubation wascontinued for 4 hours after which time 200 μL of each of the cultureswas assayed for isoprene accumulation with a modified head space assay(1 hour accumulation at 30° C. with shaking at 500 rpm). An additionalisoprene assay was conducted by a 12 hour incubation of the assay glassblock prior to GCMS analysis. Incubation of the induced cultures wascontinued overnight and 200 μL aliquots were again assayed for isopreneproduction (1 hour, 30 deg, 500 rpm Shel-Lab shaker) the followingmorning. Analysis of these cultures showed the production of significantlevels of isoprene. The highest levels of isoprene were observed in theculture which was seeded at 1/10 dilution from the overnight starterculture after it had been incubated and induced overnight. This resultsuggests that this culture continued to grow and increase in celldensity. These results are shown in FIG. 70B. Cell density could not bemeasured directly because the fatty acid suspension had a turbidappearance. Cell density of this culture was therefore determined byplating an aliquot of the culture and showed 8×10⁷ colony forming units.This corresponds approximately to an OD₆₀₀ of 0.1. Nevertheless, thisculture provided significant isoprene production; no isoprene isobserved for similar strains without the pathway described in thisexample.

Example 28 Expression of Isoprene-Synthase from Plant in Streptomycessp.

The gene for isoprene synthase Kudzu was obtained from plasmidpJ201:19813. Plasmid pJ201:19813 encodes isoprene synthase from Pueraialobata (Kudzu plant) and was codon-optimized for Pseudomonasfluorescens, Pseudomonas putida, Rhodopseudomonas palustris andCorynebacterium (FIGS. 79A-79C (SEQ ID NO:123)). Digestion of plasmidpJ201:19813 with restriction enzymes NdeI and BamHI liberated geneiso19813 that was ligated into the Streptomyces-E. coli shuttle vectorpUWL201PW (Doumith et al., Mol. Gen. Genet. 264: 477-485, 2000; FIG. 71)to generate pUWL201_iso. Successful cloning was verified by restrictionanalysis of pUWL201_iso. Expression of isoprene synthase iso19813 wasunder control of the erm-promoter which allows for constitutiveexpression in Streptomycetes species, but not for expression in E. coli.

PUWL201PW (no insert) and pUWL201 iso were introduced in Streptomycesalbus J1074 (Sanchez et al., Chem. Biol. 9:519-531, 2002) bytransformation of protoplasts as described by Hopwood et al., The Johninnes foundation, Norwich, 1985.

A 200 μl aliquot of protoplast suspensions was transformed with 1.9 ngpUWL201PW or 2.9 ng pUWL201_iso. After incubation overnight at 28° C. onnon-selective R5-agarplates, positive transformants were selected byfurther incubation for 4 days in R3-overlay agar containing thiostrepton(250 μg/ml). Thiostrepton resistant transformants were examined forpresence of the pUWL-plasmids by plasmid preparation using Plasmid MiniKit (Qiagen). Prepared plasmid DNA was reintroduced in E. coli DH5α togenerate sufficient amounts of plasmid DNA to be analyzed by restrictionanalysis. Positive transformants were selected on ampicillin-containingL-agar plates and insert analysis was done by digestion of plasmid DNAwith NdeI and BamHI endonucleases. Isoprene synthase was identified as a1.7 kb fragment in positive pUWL201 iso clones while in the controlstrains (pUWL201PW) no such fragment was observed.

Wild type strain and transformants of S. albus containing controlplasmid pUWL201PW or isoprene synthase encoding pUWL201 iso wereanalyzed for isoprene formation. Strains were cultivated in duplicate onsolid media (tryptic soy broth agar, TSB; 2.5 ml) in presence or absenceof thiostrepton (200 ng/ml) and incubated for 4 days at 28° C. in sealedhead-space vials (total volume 20 ml). 500 μl head-space samples (endpoint measurements) were analyzed by GC-MS in SIM-mode and isoprene wasidentified according to reference retention times and molecular masses(67 m/z). Isoprene present in head-space samples was quantified bypreviously generated calibration curves. While wild-type S. albus andcontrol strains harboring pUWL201PW produced isoprene in concentrationsslightly higher than the detection limit (0.04-0.07 ppm), S. albusharboring pUWL201 iso produced isoprene in at least tenfold excesscompared to controls (0.75 ppm; FIG. 72). The results demonstratesuccessful expression of plant-derived isoprene synthase in aprokaryotic organism of the Actinomycetes group.

Example 29 Recovery of Bioisoprene™

Bioisoprene™ was recovered from a set of four 14-L scale fermentationsin a two-step operation involving stripping of isoprene from thefermentation off-gas stream by adsorption to activated carbon, followedby off-line steam desorption and condensation to give liquidBioisoprene™ (FIGS. 166A and 166B). The total amount of Bioisoprene™produced by the four fermentors was 1150 g (16.9 mol), of which 953 g(14 mol, 83%) was adsorbed by the carbon filters. Following the steamdesorption/condensation step, the amount of liquid Bioisoprene™recovered was 810 g, corresponding to an overall recovery yield of 70%.The recovered Bioisoprene™ was analyzed for the presence of impurities.

Analysis and Impurity Profile of Bioisoprene™ Liquid

Recovered Bioisoprene™ liquid was analyzed by GC/MS and gaschromatography/flame ionization detection (GC/FID) to determine thenature and levels of impurities. The product was determined to be >99.5%pure and contained several dominant impurities in addition to many minorcomponents. The GC/FID chromatogram is depicted in FIG. 167, and thetypical levels of impurities are shown in Table 19. The impurity profilewas similar to other Bioisoprene™ batches produced on this scale.

TABLE 19 Summary of the nature and levels of impurities seen in severalbatches of Bioisoprene ™. Retention Time (min) Compound GC/MS GC/FIDConc. Range Ethanol 1.59 11.89 <50 ppm Acetone 1.624 12.673 <100 ppmMethacrolein 1.851 15.369 <200 ppm Methyl vinyl ketone 1.923 16.333 <20ppm Ethyl acetate 2.037 17.145 100 to 800 ppm 3-Methyl-1,3- 2.27 18.87550 to 500 ppm pentadiene Methyl vinyl oxirane 2.548 19.931 <100 ppmIsoprenol 2.962 21.583 <500 ppm 3-methyl-1-butanol 2.99 21.783 <50 ppm3-hexen-1-ol 4.019 24.819 <100 ppm Isopentenyl acetate 4.466 25.733 200to 1000 ppm 3-hexen-1-yl acetate 5.339 27.223 <400 ppm limonene 5.71527.971 <500 ppm Other cyclics 5.50-6.50 27.5-28.0 <200 ppmPurification of Bioisoprene™ by Treatment with Adsorbents

Adsorbents are widely used by industry for the removal of traceimpurities from hydrocarbon feedstocks. Suitable adsorbents includezeolite, alumina and silica-based materials. Bioisoprene™ can besubstantially purified by passage over silica gel, and to a lesserextent with alumina. FIG. 168 shows the GC/FID chromatograms of aBioisoprene™ sample before (A) and after treatment with alumina (B) orsilica (C). The Selexsorb™ adsorbent products from BASF is one of theadsorbents of choice for the removal of polar impurities fromBioisoprene™ Specifically, the Selexsorb CD and CDX products arepreferred given their proven utility for removal of polar impuritiesfrom isoprene and butadiene feedstocks.

Example 30 Chemical Transformations of Bioisoprene™

Chemicals and solvents were used as received from Sigma Aldrich Corp(WI, USA). BioIsoprene™ was produced by fermentation of E. coli BL21strains expressing Isoprene synthase and a heterologous mevalonic acid(MVA) isoprene precursor biosynthetic pathway. Bioisoprene was recoveredfrom fermentation off-gas by adsorption to activated carbon, followed bysteam desorption and condensation to obtain crude, liquid Bioisoprene.Bioisoprene was purified by fractional distillation immediately beforeuse.

¹H NMR Analysis

Proton (¹H) nuclear magnetic resonance (NMR) spectra were recorded on aVarian VNMRS 500 MHz NMR system. All NMR spectra are referenced totetramethylsilane (TMS, 0 ppm) or chloroform (CHCl₃, 7.26 ppm) and peakfrequencies are recorded in ppm unless otherwise specified. Samples wererun in either deuterated chloroform (CDCl₃) or methanol (CD₃OD).

GC/MS Analysis

The analysis was performed using an Agilent 6890 GC/MS system interfacedwith a CTC Analytics (Switzerland) CombiPAL autosampler operating inheadspace mode. An Agilent HP-5MS GC/MS column (30 m×0.25 mm; 0.25 μmfilm thickness) was used for separation of analytes. The autosampler wasset up to inject 1 μL of a liquid sample from a 10 μL liquids syringe.The GC/MS method utilized helium as the carrier gas at a flow of 1mL/minute. The injection port was held at 250° C. with a split ratio of100:1. The oven program began at 50° C. for 2 minutes, increasing to225° C. at a rate of 25° C./min. followed by a 1 minute hold for a totalrun time of 10 minutes. The Agilent 5793N mass selective detector wasrun in scan mode from m/z 29 to 500. A solvent delay of 1.5 minutes wasemployed. Under these conditions isoprene (2-methyl-1,3-butadiene) wasobserved to elute at 0.675 minutes.

GC/FID Analysis

The analysis was performed using an Agilent 6890 GC/FID systeminterfaced with a CTC Analytics (Switzerland) CombiPAL autosampleroperating in liquids mode. An Agilent DB-Petro GC column (100 m×0.25 mm;0.50 μm film thickness) was used for separation of analytes. Theautosampler was set up to inject 1 μL of a liquid sample from a 10 μLliquids syringe. The GC/FID method utilized helium as the carrier gas ata flow of 1 mL/minute. The injection port was held at 200° C. with asplit ratio of 50:1. The oven program began at 50° C. for 15 minutes,increasing to 250° C. at a rate of 25° C./min. followed by a 10 minutehold for a total run time of 33 minutes. The FID detector was held at280° C. in Constant makeup mode with a hydrogen flow of 35 mL/min andair flow of 250 mL/min. Under these conditions isoprene(2-methyl-1,3-butadiene) was observed to elute at 13.54 minutes.

I. Preparation of Cyclic Dimers of Isoprene through Thermal Diels-AlderCycloaddition

Isoprene (1.02 g, 0.015 mole) was heated at 150° C. for 24 hours in thepresence of 2,6-di-tert-butyl-4-methylphenol (0.165 g, 0.05 mole) actingas an inhibitor of thermal polymerization. The reaction was carried outin a sealed thick-walled glass reaction vessel that was dried in thevacuum oven for 24 hours prior to the reaction. The reaction mixture wasstirred with a magnetic stirrer. The progress of the reaction wasmonitored by gas chromatography with mass-spectrometer detector (GC-MS).After the reaction was finished, the resulting mixture of isomers waspurified using silica gel chromatography with hexane as the eluent.Following concentration on a rotary vacuum evaporator the product wascharacterized by GC-MS and ¹H-NMR. GC-MS: product A: 5.17, 5.19 min;product B: 5.72, 5.74 min; product C, 6.11 min. ¹H-NMR (CDCl₃) d: 5.8(m); 5.4 (m); 4.95 (m); 2.4-1.2 (m).

II. Preparation of Isoprene Oligomers through Pd-CatalyzedOligomerization

Isoprene (2.03 g, 0.03 mole) was mixed with isopropanol (1.79 g, 0.03mole) in a sealed thick-walled glass reaction vessel. Prior to thereaction the glass chamber was dried in a vacuum oven for 24 hours.Transfer of all reagents was done under inert nitrogen atmosphere.Palladium acetylacetonate (0.55 mg, 0.06 mmole) and triphenylphosphine(1.49 mg, 0.19 mmol) were added to the reaction mixture. The reactionchamber was then heated to 100° C. for 24 hours with mixing provided viamagnetic stirrer. The course of the reaction was analyzed by GC-MS. Oncethe reaction was finished the products were isolated by silica gelcolumn chromatography using hexane as the eluent. The final productswere characterized by GC-MS and ¹H-NMR spectroscopy. GC-MS: product A:5.54 min; product B: 6.6 min; product C, 6.85 min.

III. Hydrogenation of Unsaturated Compounds

The mixture of isomers obtained in Example 2 (1.36 g, 0.01 mole) is putin a glass chamber equipped with magnetic stirrer and containing ahydrogenation catalyst (Pd/C, 5 wt %) (0.21 g, 0.1 mmol). All glasswareis vacuum dried prior to carrying out the experiments. Hydrogen gas isintroduced into the system and the pressure is kept at 3 atm. Afterseveral hours the reaction mixture Pd/C is filtered from the reactionmixture and the products are separated using silica gel chromatography.Final analysis is done using GC-MS and NMR.

IV. Preparation of Ethoxylated Derivatives of Isoprene

Isoprene (0.982 g, 0.014 mole) was mixed with absolute ethanol (0.665 g,0.014 mol) in a thick glass wall chamber equipped with magnetic stirrer.A catalytic amount of concentrated sulfuric acid was added to thereaction mixture, followed by stirring overnight at 85° C. The progressof the reaction was monitored by GC-MS. After 16 hours of heating theGC-MS trace revealed the presence of a mixture of isomers with followingretention times: product A: 2.66 min m/z⁺=99; product B: 3.88 m/z⁺=99,114; product C, 5.41 min, m/z⁺=87, 99, 114.

V. Conversion of Isoprene to C10 Cyclic Dimers Using a RutheniumCatalyst

The conversion of isoprene into C10 cyclic dimers has been achieved inexcellent yield using a ruthenium catalyst to a mixture ofdimethyl-cyclooctadienes (Itoh, Kenji; Masuda, Katsuyuki; Fukahori,Takahiko; Nakano, Katsumasa; Aoki, Katsuyuki; Nagashima, Hideo,Organometallics (1994), 13(3), 1020-9.) Conversions higher than 95% arereported at very moderate temperatures (as low as 60° C.). The catalyst,a pentamethylcyclopentadienyl-based ruthenium organometallic compound,can be prepared in two easy steps using ruthenium chloride (RuCl₃) andpentamethylcyclopentadiene (C₅Me₅H) as starting materials. For thedimerization reaction the catalyst is activated with silver triflate(AgOTf) to produce the active species, but other activators could beused as well.

VI. Conversion of Isoprene to C15 Trimers Using a Chromium Catalyst

Conversion of isoprene into the C15 trimers can be carried out using achromium catalyst with a P—N—P ligand, N,N-bis(diarylphosphino)amine.The catalyst is prepared in situ and it is activated using MAO, asdescribed in Bowen, L.; Charernsuk, M.; Wass, D. F. Chem. Commun. (2007)2835-2837. The reported conversion of isoprene into the C15 trimers,consisting in a mixture (3:1) of linear and cyclic products, is as highas 95% at moderate temperatures (70° C.). Tetramers are identified asthe major other product in every case.

VII. Hydrogenation of Isoprene

Isoprene (10 mL of a 10% solution in absolute ethanol (v/v)) washydrogenated to 2-methylbutane (isopentane) in a continuous manner usingan H-cube hydrogenation instrument (ThalesNano, Princeton, N.J.,U.S.A.). The isoprene solution was pumped at 0.5 mL/min through a 10%Pd/C catalyst cartridge held at 70° C. Hydrogen gas was introduced using“full mode” at 1 atm pressure. The product was collected and analyzed by¹H NMR and GC/FID which confirmed the conversion of isoprene to2-methylbutane in over 90% yield, in addition to minor amounts ofpartially hydrogenated mono-olefins. ¹H NMR (500 MHz, CDCl₃): δ0.8 (m,9H, CH₃); 1.12 (m, 2H, CH₂); 1.37 (m, 1H, CH). GC/FID: 2-methylbutane;retention time=12.69 minutes.

VIII. Partial Hydrogenation of Isoprene

BioIsoprene™ product (50 mL, 0.5 mol) was mixed with toluene (200 mL)and partially hydrogenated over a 5% Pd/C catalyst on an Midi-Cubehydrogenation instrument (ThalesNano, Budapest, Hungary) at 40° C. and 5bar hydrogen pressure. Substrate flow rate was 10 mL/min and hydrogenwas delivered at 125 mL/min (5 mmol/min). The product stream wasrecycled through the instrument for a period of 2 hours after which timean aliquot of the product was analyzed by GC/MS and GC/FID which showedthat the majority of the starting material had been converted to amixture of isoamylenes (2-methyl-1-butene, 2-methyl-2-butene and3-methyl-1-butene), in addition to isopentane and some unreactedisoprene (FIG. 170).

IX. Selective Hydrogenation of BioIsoprene™ Product

BioIsoprene™ product is selectively hydrogenated under the conditionscited in the above example using an eggshell Pd/d-Al₂O₃ catalyst givinga mixture of isoamylenes where 2-methyl-2-butene is the dominant productaccounting for >50% of the total isoamylenes and 3-methyl-1-butene isthe minor product accounting for <25% of the total isoamylene productsas determined by GC/MS analysis. The amount of isopentane and residualisoprene account for <10% of the total product stream. A similar resultis obtained when a sulfided palladium on carbon catalyst is used toperform the reaction.

X. Partial Hydrogenation of BioIsoprene™ Product in the Gas Phase

A dry gas stream containing BioIsoprene™ product is mixed with a slightexcess of hydrogen gas (mol/mol) and the gaseous mixture passed over aheterogenous hydrogenation catalyst, such as a Group IB-promotedpalladium catalyst with high pore volume as described in US Pat. Appl.20090203520, to produce a mixture of isoamylenes and one or moreimpurities derived from the fermentation process from which theBioIsoprene™ product was originally derived. The conversion is carriedout at pressures ranging from 0.5 to 200 bar, and temperatures from 0°C. to 200° C.

XI. Dimerization of Isoamylenes with a Solid Acid Catalyst

2-Methyl-2-butene (1.5 mL) and toluene (4 mL) were stirred at roomtemperature with Amberlyst 15 acid resin (186 mg) for 12 h at roomtemperature. An aliquot (500 uL) was removed from the reaction mixtureand transferred to a GC vial. Analysis of the mixture was performed byGC/MS (FIG. 171) and revealed the partial conversion to C10 dimers(diisoamlylenes) suitable as BioIsofuel™ components.

The products of the dimerization reaction (diisoamylenes, C10 dimate)are optionally fully hydrogenated under conditions described in Example30, part VII, or through conditions known in the art for thehydrogenation of olefins to isoparaffins [for example, see Marichonna(2001)].

XII. Oligomerization of BioIsoprene™ Product with a Solid Acid Catalyst

A mixture of BioIsoprene™ monomer, 2-methyl-2-butene (1.5 mL) andtoluene (4 mL) was stirred at room temperature with Amberlyst 15 acidresin (186 mg) for 12 h at room temperature. An aliquot (500 uL) wasremoved from the reaction mixture and transferred to a GC vial. Analysisof the mixture was performed by GC/MS (FIG. 172) and revealed a complexmixture of products consisting of isoprene, linear, cyclic and aromaticC10, C15 and higher oligomers.

XIII. Continuous Oligomerization of BioIsoprene™ Product with a SolidAcid Catalyst

BioIsoprene™ monomer is continuously converted into C10 dimers and C15trimers in a dimerization reactor containing Amberlyst 15 ion exchangeresin or an equivalent catalyst. The BioIsoprene™ feed stream comprisesBioIsoprene™ monomer and optionally C5 derivatives of BioIsoprene™ and aco-solvent. The process is conducted at temperatures ranging from 20 to200° C. and pressures from 0.5 to 200 bar. The products of thedimerization step are fractionated in a first fractionation column toseparate unreacted isoprene from higher (>C5) oligomers. The C5 fractionis returned to the dimerization reactor and the heavy >C5 fraction isintroduced into a second fractionation column in which the desiredC10/C15 fraction is collected from the overhead stream. The bottomfraction consisting of >C15 oligomers is fed into a heavy recyclereactor containing a metathesis catalyst such as the Grubbs 2^(nd)generation catalyst. The metathesis catalyst converts a portion of thehigher oligomer fraction into lighter components by olefincross-metathesis reactions that are subsequently fed into fractionationcolumn #1 as depicted in FIG. 173.

Overall, the process results in conversion of BioIsoprene monomer intoC10 dimer and C15 trimer BioIsofuel™ precursors which are then subjectedto partial or complete hydrogenation under conditions described inexample 30, section VII. The resulting partially or fully saturatedcompounds are suitable as BioIsofuel™ compositions and as BioIsofuel™blendstocks.

Example 31 ¹³C/¹²C Isotope Analysis

¹³C analysis can be done by loading 0.5 to 1.0 mg samples into tin cupsfor carbon isotopic analysis using a Costech ECS4010 Elemental Analyzeras an inlet for a ThermoFinnigan Delta Plus XP isotope ratio massspectrometer. Samples are dropped into a cobaltous/cobaltic oxidecombustion reactor at 1020° C. with combustion gases being passed in ahelium stream at 85 mL/min through a copper reactor (650° C.) to convertNO_(x) to N₂. CO₂ and N₂ are separated using a 3-m 5 Å molecular sievecolumn. Then, ¹³C/¹²C ratios are calibrated to the VPDB scale using twolaboratory standards (Acetanilide B, −29.52±0.02‰m and cornstarch A,−11.01±0.02‰) which have been carefully calibrated to the VPDB scale byoff-line combustion and dual-inlet analysis using the 2-standardapproach of T. B. Coplen et al, New Guidelines for d¹³C Measurements,Anal. Chem., 78, 2439-2441 (2006). The teachings of Coplen areincorporated herein by reference for the purpose of teaching thetechnique for determining d¹³C values.

U.S. Provisional Patent Application No. 61/133,521 filed on Jun. 30,2008 and WO 2010/05525 A1 list δ¹³C values for feedstock and polymers ofisoprene derived from various sources, including ones listed in Table20.

TABLE 20 Sample δ¹³C Palm oil −30.00 Yeast extract −25.70 Commercialpolyisoprene from extractive distillation −23.83 Sugar from softwoodpulp −23.00 Polyisoprene from Isoprene Sample B (emulsionpolymerization) −19.67 Invert Sugar −15.37 Polyisoprene from IsopreneSample A (Neodymium catalyst) −14.85 Glucose from bagasse −13.00 Glucosefrom corn stover −11.20 Cornstarch −11.10 Glucose −10.73

Example 32 Exemplary Fuel Properties

Table 21 lists fuel properties of certain compounds that can be madefrom isoprene using methods described herein.

TABLE 21 Lower Higher Vapor Heating Heating Boiling pressure ΔHc ValueValue MW point Density (Torr, (kcal/ (kBtu/ (kBtu/ Octane Formula(g/mol) (° C.) (g/mol³) 25° C.)* mol) gal) gal) (Cetane) a-LimoneneC₁₀H₁₆

136.23 177 0.8477 1.54 1474 130.6 137.9  88 1-Methyl-4- isopropyl-cyclohexane C₁₀H₂₀

140.27 170.2 0.8060 2.16 1561 126.5 134.9  75 2,7-dimethyl-, (4E)-2,4,6-Octatriene C₁₀H₁₆

136.23 186.4* 0.782 0.915 1481 121.1 127.8 110 2,7-dimethyl- octaneC₁₀H₂₂

142.28 160 0.728 3.35 1620 116.5 124.7  97 3,7-dimethyl- 1,5-cyclo-octadiene C₁₀H₁₆

136.23 182.7* 0.860 1.09 1490 134.0 141.4  95 1,5-Dimethyl- cyclooctene(BIF-10) C₁₀H₁₈

138.25 178.5* 0.830 1.33 1545 131.6 139.4  90 1,5-Dimethyl cyclooctaneC₁₀H₂₀

140.27 158.5 0.800 1.39 1560 125.4 133.8  85 2,6,11- Trimethyl- dodecaneC₁₅H₃₂

212.41 247.8* 0.766 0.0396 2400 121.7 130.1 (65) Cyclo- dodecane, 1,4,8-trimethyl C₁₅H₃₀

210.40 278.0* 0.850 7.38E−3 2380 135.7 144.6 (40) BIF-5 C₁₅H_(31.5) 3:1mixture of linear and cyclic C15 211.9 255.4 0.787 0.0315 2395 125.2133.7 (58.8)

The headings provided herein are not limitations of the various aspectsor embodiments of the invention which can be had by reference to thespecification as a whole.

All publications, patent applications, and patents cited in thisspecification are herein incorporated by reference as if each individualpublication, patent application, or patent were specifically andindividually indicated to be incorporated by reference. In particular,all publications cited herein are expressly incorporated herein byreference for the purpose of describing and disclosing compositions andmethodologies which might be used in connection with the invention.Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

APPENDIX 1 Exemplary 1-deoxy-D-xylulose-5-phosphate synthase nucleicacids and polypeptides ATH: AT3G21500(DXPS1) AT4G15560(CLA1) HIQ:CGSHiGG_01080 AT5G11380(DXPS3) HDU: HD0441(dxs) OSA: 4338768 43400904342614 HSO: HS_0905(dxs) CME: CMF089C PMU: PM0532(dxs) PFA: MAL13P1.186MSU: MS1059(dxs) TAN: TA20470 APL: APL_0207(dxs) TPV: TP01_0516 XFA:XF2249 ECO: b0420(dxs) XFT: PD1293(dxs) ECJ: JW0410(dxs) XCC:XCC2434(dxs) ECE: Z0523(dxs) XCB: XC_1678 ECS: ECs0474 XCV: XCV2764(dxs)ECC: c0531(dxs) XAC: XAC2565(dxs) ECI: UTI89_C0443(dxs) XOO:XOO2017(dxs) ECP: ECP_0479 XOM: XOO_1900(XOO1900) ECV: APECO1_1590(dxs)VCH: VC0889 ECW: EcE24377A_0451(dxs) VVU: VV1_0315 ECX: EcHS_A0491 VVY:VV0868 STY: STY0461(dxs) VPA: VP0686 STT: t2441(dxs) VFI: VF0711 SPT:SPA2301(dxs) PPR: PBPRA0805 SEC: SC0463(dxs) PAE: PA4044(dxs) STM:STM0422(dxs) PAU: PA14_11550(dxs) YPE: YPO3177(dxs) PAP: PSPA7_1057(dxs)YPK: y1008(dxs) PPU: PP_0527(dxs) YPM: YP_0754(dxs) PST: PSPTO_0698(dxs)YPA: YPA_2671 PSB: Psyr_0604 YPN: YPN_0911 PSP: PSPPH_0599(dxs) YPP:YPDSF_2812 PFL: PFL_5510(dxs) YPS: YPTB0939(dxs) PFO: Pfl_5007 YPI:YpsIP31758_3112(dxs) PEN: PSEEN0600(dxs) SFL: SF0357(dxs) PMY: Pmen_3844SFX: S0365(dxs) PAR: Psyc_0221(dxs) SFV: SFV_0385(dxs) PCR: Pcryo_0245SSN: SSON_0397(dxs) ACI: ACIAD3247(dxs) SBO: SBO_0314(dxs) SON:SO_1525(dxs) SDY: SDY_0310(dxs) SDN: Sden_2571 ECA: ECA1131(dxs) SFR:Sfri_2790 PLU: plu3887(dxs) SAZ: Sama_2436 BUC: BU464(dxs) SBL:Sbal_1357 BAS: BUsg448(dxs) SLO: Shew_2771 WBR: WGLp144(dxs) SHE:Shewmr4_2731 SGL: SG0656 SHM: Shewmr7_2804 KPN: KPN_00372(dxs) SHN:Shewana3_2901 BFL: Bfl238(dxs) SHW: Sputw3181_2831 BPN: BPEN_244(dxs)ILO: IL2138(dxs) HIN: HI1439(dxs) CPS: CPS_1088(dxs) HIT: NTHI1691(dxs)PHA: PSHAa2366(dxs) HIP: CGSHiEE_04795 PAT: Patl_1319 SDE: Sde_3381 HAR:HEAR0279(dxs) PIN: Ping_2240 MMS: mma_0331 MAQ: Maqu_2438 NEU:NE1161(dxs) MCA: MCA0817(dxs) NET: Neut_1501 FTU: FTT1018c(dxs) NMU:Nmul_A0236 FTF: FTF1018c(dxs) EBA: ebA4439(dxs) FTW: FTW_0925(dxs) AZO:azo1198(dxs) FTL: FTL_1072 DAR: Daro_3061 FTH: FTH_1047(dxs) TBD:Tbd_0879 FTA: FTA_1131(dxs) MFA: Mfla_2133 FTN: FTN_0896(dxs) HPY:HP0354(dxs) NOC: Noc_1743 HPJ: jhp0328(dxs) AEH: Mlg_1381 HPA:HPAG1_0349 HCH: HCH_05866(dxs) HHE: HH0608(dxs) CSA: Csal_0099 HAC:Hac_0968(dxs) ABO: ABO_2166(dxs) WSU: WS1996 AHA: AHA_3321(dxs) TDN:Tmden_0475 BCI: BCI_0275(dxs) CJE: Cj0321(dxs) RMA: Rmag_0386 CJR:CJE0366(dxs) VOK: COSY_0360(dxs) CJJ: CJJ81176_0343(dxs) NME: NMB1867CJU: C8J_0298(dxs) NMA: NMA0589(dxs) CJD: JJD26997_1642(dxs) NMC:NMC0352(dxs) CFF: CFF8240_0264(dxs) NGO: NGO0036 CCV: CCV52592_1671(dxs)CCV52592_1722 CVI: CV_2692(dxs) CHA: CHAB381_1297(dxs) RSO: RSc2221(dxs)CCO: CCC13826_1594(dxs) REU: Reut_A0882 ABU: Abu_2139(dxs) REH:H16_A2732(dxs) NIS: NIS_0391(dxs) RME: Rmet_2615 SUN: SUN_2055(dxs) BMA:BMAA0330(dxs) GSU: GSU0686(dxs-1) GSU1764(dxs-2) BMV: BMASAVP1_1512(dxs)GME: Gmet_1934 Gmet_2822 BML: BMA10299_1706(dxs) PCA: Pcar_1667 BMN:BMA10247_A0364(dxs) PPD: Ppro_1191 Ppro_2403 BXE: Bxe_B2827 DVU:DVU1350(dxs) BUR: Bcep18194_B2211 DVL: Dvul_1718 BCN: Bcen_4486 DDE:Dde_2200 BCH: Bcen2424_3879 LIP: LI0408(dsx) BAM: Bamb_3250 DPS: DP2700BPS: BPSS1762(dxs) ADE: Adeh_1097 BPM: BURPS1710b_A0842(dxs) MXA:MXAN_4643(dxs) BPL: BURPS1106A_A2392(dxs) SAT: SYN_02456 BPD:BURPS668_A2534(dxs) SFU: Sfum_1418 BTE: BTH_II0614(dxs) PUB:SAR11_0611(dxs) BPE: BP2798(dxs) MLO: mlr7474 BPA: BPP2464(dxs) MES:Meso_0735 BBR: BB1912(dxs) SME: SMc00972(dxs) RFR: Rfer_2875 ATU:Atu0745(dxs) POL: Bpro_1747 ATC: AGR_C_1351 PNA: Pnap_1501 RET:RHE_CH00913(dxs) AJS: Ajs_1038 RLE: RL0973(dxs) MPT: Mpe_A2631 BME:BMEI1498 BMF: BAB1_0462(dxs) GKA: GK2392 BMS: BR0436(dxs) GTN: GTNG_2322BMB: BruAb1_0458(dxs) LMO: lmo1365(tktB) BOV: BOV_0443(dxs) LMF:LMOf2365_1382(dxs) BJA: bll2651(dxs) LIN: lin1402(tktB) BRA:BRADO2161(dxs) LWE: lwe1380(tktB) BBT: BBta_2479(dxs) LLA: L108911(dxsA)L123365(dxsB) RPA: RPA0952(dxs) LLC: LACR_1572 LACR_1843 RPB: RPB_4460LLM: llmg_0749(dxsB) RPC: RPC_1149 SAK: SAK_0263 RPD: RPD_4305 LPL:lp_2610(dxs) RPE: RPE_1067 LJO: LJ0406 NWI: Nwi_0633 LAC: LBA0356 NHA:Nham_0778 LSL: LSL_0209(dxs) BHE: BH04350(dxs) LGA: LGAS_0350 BQU:BQ03540(dxs) STH: STH1842 BBK: BARBAKC583_0400(dxs) CAC: CAC2077CA_P0106(dxs) CCR: CC_2068 CPE: CPE1819 SIL: SPO0247(dxs) CPF:CPF_2073(dxs) SIT: TM1040_2920 CPR: CPR_1787(dxs) RSP: RSP_0254(dxsA)RSP_1134(dxs) CTC: CTC01575 JAN: Jann_0088 Jann_0170 CNO: NT01CX_1983RDE: RD1_0101(dxs) RD1_0548(dxs) CTH: Cthe_0828 MMR: Mmar10_0849 CDF:CD1207(dxs) HNE: HNE_1838(dxs) CBO: CBO1881(dxs) ZMO: ZMO1234(dxs)ZMO1598(dxs) CBA: CLB_1818(dxs) NAR: Saro_0161 CBH: CLC_1825(dxs) SAL:Sala_2354 CBF: CLI_1945(dxs) ELI: ELI_12520 CKL: CKL_1231(dxs) GOX:GOX0252 CHY: CHY_1985(dxs) GBE: GbCGDNIH1_0221 GbCGDNIH1_2404 DSY:DSY2348 RRU: Rru_A0054 Rru_A2619 DRM: Dred_1078 MAG: amb2904 PTH:PTH_1196(dxs) MGM: Mmc1_1048 SWO: Swol_0582 SUS: Acid_1783 CSC:Csac_1853 BSU: BG11715(dxs) TTE: TTE1298(dxs) BHA: BH2779 MTA: Moth_1511BAN: BA4400(dxs) MPE: MYPE730 BAR: GBAA4400(dxs) MGA: MGA_1268(dxs) BAA:BA_4853 MTU: Rv2682c(dxs1) Rv3379c(dxs2) BAT: BAS4081 MTC: MT2756(dxs)BCE: BC4176(dxs) MBO: Mb2701c(dxs1) Mb3413c(dxs2) BCA: BCE_4249(dxs)MLE: ML1038(dxs) BCZ: BCZK3930(dxs) MPA: MAP2803c(dxs) BTK:BT9727_3919(dxs) MAV: MAV_3577(dxs) BTL: BALH_3785(dxs) MSM:MSMEG_2776(dxs) BLI: BL01523(dxs) MMC: Mmcs_2208 BLD: BLi02598(dxs) CGL:NCgl1827(cgl1902) BCL: ABC2462(dxs) CGB: cg2083(dxs) BAY: RBAM_022600CEF: CE1796 BPU: BPUM_2159 CDI: DIP1397(dxs) CJK: jk1078(dxs) AVA:Ava_4532 NFA: nfa37410(dxs) PMA: Pro0928(dxs) RHA: RHA1_ro06843 PMM:PMM0907(Dxs) SCO: SCO6013(SC1C3.01) SCO6768(SC6A5.17) PMT: PMT0685(dxs)SMA: SAV1646(dxs1) SAV2244(dxs2) PMN: PMN2A_0300 TWH: TWT484 PMI:PMT9312_0893 TWS: TW280(Dxs) PMB: A9601_09541(dxs) LXX: Lxx10450(dxs)PMC: P9515_09901(dxs) CMI: CMM_1660(dxsA) PMF: P9303_15371(dxs) AAU:AAur_1790(dxs) PMG: P9301_09521(dxs) PAC: PPA1062 PMH: P9215_09851 TFU:Tfu_1917 PMJ: P9211_08521 FRA: Francci3_1326 PME: NATL1_09721(dxs) FAL:FRAAL2088(dxs) TER: Tery_3042 ACE: Acel_1393 BTH: BT_1403 BT_4099 SEN:SACE_1815(dxs) SACE_4351 BFR: BF0873 BF4306 BLO: BL1132(dxs) BFS:BF0796(dxs) BF4114 BAD: BAD_0513(dxs) PGI: PG2217(dxs) FNU: FN1208FN1464 CHU: CHU_3643(dxs) RBA: RB2143(dxs) GFO: GFO_3470(dxs) CTR:CT331(dxs) FPS: FP0279(dxs) CTA: CTA_0359(dxs) CTE: CT0337(dxs) CMU:TC0608 CPH: Cpha266_0671 CPN: CPn1060(tktB_2) PVI: Cvib_0498 CPA: CP0790PLT: Plut_0450 CPJ: CPj1060(tktB_2) DET: DET0745(dxs) CPT: CpB1102 DEH:cbdb_A720(dxs) CCA: CCA00304(dxs) DRA: DR_1475 CAB: CAB301(dxs) DGE:Dgeo_0994 CFE: CF0699(dxs) TTH: TTC1614 PCU: pc0619(dxs) TTJ: TTHA0006TPA: TP0824 AAE: aq_881 TDE: TDE1910(dxs) TMA: TM1770 LIL: LA3285(dxs)PMO: Pmob_1001 LIC: LIC10863(dxs) LBJ: LBJ_0917(dxs) LBL: LBL_0932(dxs)SYN: sll1945(dxs) SYW: SYNW1292(Dxs) SYC: syc1087_c(dxs) SYF:Synpcc7942_0430 SYD: Syncc9605_1430 SYE: Syncc9902_1069 SYG:sync_1410(dxs) SYR: SynRCC307_1390(dxs) SYX: SynWH7803_1223(dxs) CYA:CYA_1701(dxs) CYB: CYB_1983(dxs) TEL: tll0623 GVI: gll0194 ANA: alr0599Exemplary acetyl-CoA-acetyltransferase nucleic acids and polypeptidesHSA: 38(ACAT1) 39(ACAT2) STM: STM3019(yqeF) PTR: 451528(ACAT1) SFL:SF2854(yqeF) MCC: 707653(ACAT1) 708750(ACAT2) SFX: S3052(yqeF) MMU:110446(Acat1) 110460(Acat2) SFV: SFV_2922(yqeF) RNO: 25014(Acat1) SSN:SSON_2283(atoB) SSON_3004(yqeF) CFA: 484063(ACAT2) 489421(ACAT1) SBO:SBO_2736(yqeF) GGA: 418968(ACAT1) 421587(RCJMB04_34i5) ECA:ECA1282(atoB) XLA: 379569(MGC69098) 414622(MGC81403) ENT: Ent638_3299414639(MGC81256) SPE: Spro_0592 444457(MGC83664) HIT: NTHI0932(atoB)XTR: 394562(acat2) XCC: XCC1297(atoB) DRE: 30643(acat2) XCB: XC_2943SPU: 759502(LOC759502) XCV: XCV1401(thlA) DME: Dmel_CG10932 Dmel_CG9149XAC: XAC1348(atoB) CEL: T02G5.4 T02G5.7 T02G5.8(kat-1) XOO:XOO1881(atoB) ATH: AT5G48230(ACAT2/EMB1276) XOM: XOO_1778(XOO1778) OSA:4326136 4346520 VCH: VCA0690 CME: CMA042C CME087C VCO: VC0395_0630 SCE:YPL028W(ERG10) VVU: VV2_0494 VV2_0741 AGO: AGOS_ADR165C VVY: VVA1043VVA1210 PIC: PICST_31707(ERG10) VPA: VPA0620 VPA1123 VPA1204 CAL:CaO19.1591(erg10) PPR: PBPRB1112 PBPRB1840 CGR: CAGL0L12364g PAE:PA2001(atoB) PA2553 PA3454 PA3589 SPO: SPBC215.09c PA3925 MGR: MGG_01755MGG_13499 PAU: PA14_38630(atoB) ANI: AN1409.2 PPU: PP_2051(atoB)PP_2215(fadAx) PP_3754 AFM: AFUA_6G14200 AFUA_8G04000 PP_4636 AOR:AO090103000012 AO090103000406 PPF: Pput_2009 Pput_2403 Pput_3523Pput_4498 CNE: CNC05280 PST: PSPTO_0957(phbA-1) PSPTO_3164(phbA-2) UMA:UM03571.1 PSB: Psyr_0824 Psyr_3031 DDI: DDB_0231621 PSP:PSPPH_0850(phbA1) PSPPH_2209(phbA2) PFA: PF14_0484 PFL: PFL_1478(atoB-2)PFL_2321 PFL_3066 TET: TTHERM_00091590 TTHERM_00277470 PFL_4330(atoB-2)PFL_5283 TTHERM_00926980 PFO: Pfl_1269 Pfl_1739 Pfl_2074 Pfl_2868 TCR:511003.60 PEN: PSEEN3197 PSEEN3547(fadAx) ECO: b2224(atoB)PSEEN4635(phbA) ECJ: JW2218(atoB) JW5453(yqeF) PMY: Pmen_1138 Pmen_2036Pmen_3597 ECE: Z4164(yqeF) Pmen_3662 Pmen_3820 ECS: ECs3701 PAR:Psyc_0252 Psyc_1169 ECC: c2767(atoB) c3441(yqeF) PCR: Pcryo_0278Pcryo_1236 Pcryo_1260 ECI: UTI89_C2506(atoB) UTI89_C3247(yqeF) PRW:PsycPRwf_2011 ECP: ECP_2268 ECP_2857 ACI: ACIAD0694 ACIAD1612ACIAD2516(atoB) ECV: APECO1_3662(yqeF) APECO1_4335(atoB) SON:SO_1677(atoB) APECO1_43352(atoB) SDN: Sden_1943 ECX: EcHS_A2365 SFR:Sfri_1338 Sfri_2063 STY: STY3164(yqeF) SAZ: Sama_1375 STT: t2929(yqeF)SBL: Sbal_1495 SPT: SPA2886(yqeF) SBM: Shew185_1489 SEC: SC2958(yqeF)SBN: Sbal195_1525 SLO: Shew_1667 Shew_2858 BXE: Bxe_A2273 Bxe_A2335Bxe_A2342 SPC: Sputcn32_1397 Bxe_A4255 Bxe_B0377 Bxe_B0739 SSE:Ssed_1473 Ssed_3533 Bxe_C0332 Bxe_C0574 Bxe_C0915 SPL: Spea_2783 BVI:Bcep1808_0519 Bcep1808_1717 SHE: Shewmr4_2597 Bcep1808_2877Bcep1808_3594 SHM: Shewmr7_2664 Bcep1808_4015 Bcep1808_5507Bcep1808_5644 SHN: Shewana3_2771 BUR: Bcep18194_A3629 Bcep18194_A5080SHW: Sputw3181_2704 Bcep18194_A5091 ILO: IL0872 Bcep18194_A6102Bcep18194_B0263 CPS: CPS_1605 CPS_2626 Bcep18194_B1439 PHA: PSHAa0908PSHAa1454(atoB) Bcep18194_C6652 Bcep18194_C6802 PSHAa1586(atoB)Bcep18194_C6874 PAT: Patl_2923 Bcep18194_C7118 Bcep18194_C7151 SDE:Sde_3149 Bcep18194_C7332 PIN: Ping_0659 Ping_2401 BCN: Bcen_1553Bcen_1599 Bcen_2158 Bcen_2563 MAQ: Maqu_2117 Maqu_2489 Maqu_2696Bcen_2998 Bcen_6289 Maqu_3162 BCH: Bcen2424_0542 Bcen2424_1790 CBU:CBU_0974 Bcen2424_2772 Bcen2424_5368 LPN: lpg1825(atoB) Bcen2424_6232Bcen2424_6276 LPF: lpl1789 BAM: Bamb_0447 Bamb_1728 Bamb_2824 LPP:lpp1788 Bamb_4717 Bamb_5771 Bamb_5969 NOC: Noc_1891 BPS: BPSL1426BPSL1535(phbA) BPSL1540 AEH: Mlg_0688 Mlg_2706 BPM:BURPS1710b_2325(bktB) HHA: Hhal_1685 BURPS1710b_2330(phbA) HCH:HCH_05299 BURPS1710b_2453(atoB-2) CSA: Csal_0301 Csal_3068 BPL:BURPS1106A_2197(bktB) ABO: ABO_0648(fadAx) BURPS1106A_2202(phbA) MMW:Mmwyl1_0073 Mmwyl1_3021 Mmwyl1_3053 BPD: BURPS668_2160(bktB)BURPS668_2165(phbA) Mmwyl1_3097 Mmwyl1_4182 BTE: BTH_I2144 BTH_I2256BTH_I2261 AHA: AHA_2143(atoB) PNU: Pnuc_0927 CVI: CV_2088(atoB)CV_2790(phaA) BPE: BP0447 BP0668 BP2059 RSO: RSc0276(atoB) RSc1632(phbA)RSc1637(bktB) BPA: BPP0608 BPP1744 BPP3805 BPP4216 RSc1761(RS02948)BPP4361 REU: Reut_A0138 Reut_A1348 Reut_A1353 BBR: BB0614 BB3364 BB4250BB4804 BB4947 Reut_B4561 Reut_B4738 RFR: Rfer_0272 Rfer_1000 Rfer_1871Rfer_2273 Reut_B5587 Reut_C5943 Reut_C6062 Rfer_2561 Rfer_2594 REH:H16_A0170 H16_A0867 H16_A0868 Rfer_3839 H16_A0872 H16_A1297 POL:Bpro_1577 Bpro_2140 Bpro_3113 Bpro_4187 H16_A1438(phaA) H16_A1445(bktB)H16_A1528 PNA: Pnap_0060 Pnap_0458 Pnap_0867 Pnap_1159 H16_A1713H16_A1720 Pnap_2136 Pnap_2804 H16_A1887 H16_A2148 H16_B0380 H16_B0381AAV: Aave_0031 Aave_2478 Aave_3944 Aave_4368 H16_B0406 H16_B0662 AJS:Ajs_0014 Ajs_0124 Ajs_1931 Ajs_2073 H16_B0668 H16_B0759 H16_B1369H16_B1771 Ajs_2317 Ajs_3548 RME: Rmet_0106 Rmet_1357 Rmet_1362 Rmet_5156Ajs_3738 Ajs_3776 BMA: BMA1316 BMA1321(phbA) BMA1436 VEI: Veis_1331Veis_3818 Veis_4193 BMV: BMASAVP1_A1805(bktB) DAC: Daci_0025 Daci_0192Daci_3601 Daci_5988 BMASAVP1_A1810(phbA) MPT: Mpe_A1536 Mpe_A1776Mpe_A1869 BML: BMA10299_A0086(phbA) BMA10299_A0091 Mpe_A3367 BMN:BMA10247_1076(bktB) HAR: HEAR0577(phbA) BMA10247_1081(phbA) MMS:mma_0555 NEU: NE2262(bktB) NET: Neut_0610 RPC: RPC_0504 RPC_0636RPC_0641 RPC_0832 EBA: ebA5202 p2A409(tioL) RPC_1050 RPC_2005 AZO:azo0464(fadA1) azo0469(fadA2) azo2172(thlA) RPC_2194 RPC_2228 DAR:Daro_0098 Daro_3022 RPD: RPD_0306 RPD_0320 RPD_3105 RPD_3306 HPA:HPAG1_0675 RPE: RPE_0168 RPE_0248 RPE_3827 HAC: Hac_0958(atoB) NWI:Nwi_3060 GME: Gmet_1719 Gmet_2074 Gmet_2213 XAU: Xaut_3108 Xaut_4665Gmet_2268 Gmet_3302 CCR: CC_0510 CC_0894 CC_3462 GUR: Gura_3043 SIL:SPO0142(bktB) SPO0326(phbA) SPO0773 BBA: Bd0404(atoB) Bd2095 SPO3408DOL: Dole_0671 Dole_1778 Dole_2160 Dole_2187 SIT: TM1040_0067TM1040_2790 TM1040_3026 ADE: Adeh_0062 Adeh_2365 TM1040_3735 AFW:Anae109_0064 Anae109_1504 RSP: RSP_0745 RSP_1354 RSP_3184 MXA: MXAN_3791RSH: Rsph17029_0022 Rsph17029_2401 SAT: SYN_02642 Rsph17029_3179Rsph17029_3921 SFU: Sfum_2280 Sfum_3582 RSQ: Rsph17025_0012Rsph17025_2466 RPR: RP737 Rsph17025_2833 RCO: RC1134 RC1135 JAN:Jann_0262 Jann_0493 Jann_4050 RFE: RF_0163(paaJ) RDE: RD1_0025RD1_0201(bktB) RD1_3394(phbA) RBE: RBE_0139(paaJ) PDE: Pden_2026Pden_2663 Pden_2870 Pden_2907 RAK: A1C_05820 Pden_4811 Pden_5022 RBO:A1I_07215 DSH: Dshi_0074 Dshi_3066 Dshi_3331 RCM: A1E_04760 MMR:Mmar10_0697 PUB: SAR11_0428(thlA) HNE: HNE_2706 HNE_3065 HNE_3133 MLO:mlr3847 NAR: Saro_0809 Saro_1069 Saro_1222 Saro_2306 MES: Meso_3374Saro_2349 PLA: Plav_1573 Plav_2783 SAL: Sala_0781 Sala_1244 Sala_2896Sala_3158 SME: SMa1450 SMc03879(phbA) SWI: Swit_0632 Swit_0752 Swit_2893Swit_3602 SMD: Smed_0499 Smed_3117 Smed_5094 Swit_4887 Swit_5019Smed_5096 Swit_5309 ATU: Atu2769(atoB) Atu3475 ELI: ELI_01475 ELI_06705ELI_12035 ATC: AGR_C_5022(phbA) AGR_L_2713 GBE: GbCGDNIH1_0447 RET:RHE_CH04018(phbAch) ACR: Acry_1847 Acry_2256 RHE_PC00068(ypc00040)RHE_PF00014(phbAf) RRU: Rru_A0274 Rru_A1380 Rru_A1469 Rru_A1946 RLE:RL4621(phaA) pRL100301 pRL120369 Rru_A3387 BME: BMEI0274 BMEII0817 MAG:amb0842 BMF: BAB1_1783(phbA-1) BAB2_0790(phbA-2) MGM: Mmc1_1165 BMS:BR1772(phbA-1) BRA0448(phbA-2) ABA: Acid345_3239 BMB:BruAb1_1756(phbA-1) BruAb2_0774(phbA-2) BSU: BG11319(mmgA) BG13063(yhfS)BOV: BOV_1707(phbA-1) BHA: BH1997 BH2029 BH3801(mmgA) OAN: Oant_1130Oant_3107 Oant_3718 Oant_4020 BAN: BA3687 BA4240 BA5589 BJA:bll0226(atoB) bll3949 bll7400 bll7819 BAR: GBAA3687 GBAA4240 GBAA5589blr3724(phbA) BAA: BA_0445 BA_4172 BA_4700 BRA: BRADO0562(phbA)BRADO0983(pimB) BAT: BAS3418 BAS3932 BAS5193 BRADO3110 BRADO3134(atoB)BCE: BC3627 BC4023 BC5344 BBT: BBta_3558 BBta_3575(atoB) BBta_5147(pimB)BCA: BCE_3646 BCE_4076 BCE_5475 BBta_7072(pimB) BCZ: BCZK3329(mmgA)BCZK3780(thl) BBta_7614(phbA) BCZK5044(atoB) RPA: RPA0513(pcaF) RPA0531RPA3715(pimB) BCY: Bcer98_2722 Bcer98_3865 RPB: RPB_0509 RPB_0525RPB_1748 BTK: BT9727_3379(mmgA) BT9727_3765(thl) BTL: BALH_3262(mmgA)BALH_3642(fadA) BT9727_5028(atoB) BALH_4843(atoB) LJO: LJ1609 BLI:BL03925(mmgA) LAC: LBA0626(thiL) BLD: BLi03968(mmgA) LSA: LSA1486 BCL:ABC0345 ABC2989 ABC3617 LDB: Ldb0879 ABC3891(mmgA) LBU: LBUL_0804 BAY:RBAM_022450 LBR: LVIS_2218 BPU: BPUM_2374(yhfS) BPUM_2941 BPUM_3373 LCA:LSEI_1787 OIH: OB0676 OB0689 OB2632 OB3013 LGA: LGAS_1374 GKA: GK1658GK3397 LRE: Lreu_0052 SAU: SA0342 SA0534(vraB) EFA: EF1364 SAV: SAV0354SAV0576(vraB) OOE: OEOE_0529 SAM: MW0330 MW0531(vraB) STH: STH2913STH725 STH804 SAR: SAR0351(thl) SAR0581 CAC: CAC2873 CA_P0078(thiL) SAS:SAS0330 SAS0534 CPE: CPE2195(atoB) SAC: SACOL0426 SACOL0622(atoB) CPF:CPF_2460 SAB: SAB0304(th1) SAB0526 CPR: CPR_2170 SAA: SAUSA300_0355SAUSA300_0560(vraB) CTC: CTC00312 SAO: SAOUHSC_00336 SAOUHSC_00558 CNO:NT01CX_0538 NT01CX_0603 SAJ: SaurJH9_0402 CDF: CD1059(thlA1)CD2676(thlA2) SAH: SaurJH1_0412 CBO: CBO3200(thl) SEP: SE0346 SE2384CBE: Cbei_0411 Cbei_3630 SER: SERP0032 SERP0220 CKL: CKL_3696(thlA1)CKL_3697(thlA2) SHA: SH0510(mvaC) SH2417 CKL_3698(thlA3) SSP: SSP0325SSP2145 AMT: Amet_4630 LMO: lmo1414 AOE: Clos_0084 Clos_0258 LMF:LMOf2365_1433 CHY: CHY_1288 CHY_1355(atoB) CHY_1604 LIN: lin1453CHY_1738 LWE: lwe1431 DSY: DSY0632 DSY0639 DSY1567 DSY1710 LLA:L11745(thiL) L25946(fadA) DSY2402 DSY3302 LLC: LACR_1665 LACR_1956 DRM:Dred_0400 Dred_1491 Dred_1784 Dred_1892 LLM: llmg_0930(thiL) SWO:Swol_0308 Swol_0675 Swol_0789 Swol_1486 SPY: SPy_0140 SPy_1637(atoB)Swol_1934 Swol_2051 SPZ: M5005_Spy_0119 M5005_Spy_0432 TTE:TTE0549(paaJ) M5005_Spy_1344(atoB) MTA: Moth_1260 SPM: spyM18_0136spyM18_1645(atoB) MTU: Rv1135A Rv1323(fadA4) Rv3546(fadA5) SPG:SpyM3_0108 SpyM3_1378(atoB) MTC: MT1365(phbA) SPS: SPs0110 SPs0484 MBO:Mb1167 Mb1358(fadA4) Mb3576(fadA5) SPH: MGAS10270_Spy0121MGAS10270_Spy0433 Mb3586c(fadA6) MGAS10270_Spy1461(atoB) MBB: BCG_1197BCG_1385(fadA4) SPI: MGAS10750_Spy0124 MGAS10750_Spy0452 BCG_3610(fadA5)BCG_3620c(fadA6) MGAS10750_Spy1453(atoB) MLE: ML1158(fadA4) SPJ:MGAS2096_Spy0123 MGAS2096_Spy0451 MPA: MAP2407c(fadA3) MAP2436c(fadA4)MGAS2096_Spy1365(atoB) MAV: MAV_1544 MAV_1573 MAV_1863 SPK:MGAS9429_Spy0121 MGAS9429_Spy0431 MAV_5081 MGAS9429_Spy1339(atoB) MSM:MSMEG_2224 MSMEG_4920 SPF: SpyM50447(atoB2) MUL: MUL_0357 SPA:M6_Spy0166 M6_Spy0466 M6_Spy1390 MVA: Mvan_1976 Mvan_1988 Mvan_4305 SPB:M28_Spy0117 M28_Spy0420 Mvan_4677 Mvan_4891 M28_Spy1385(atoB) MGI:Mflv_1347 Mflv_1484 Mflv_2040 Mflv_2340 SAK: SAK_0568 Mflv_4356Mflv_4368 MMC: Mmcs_1758 Mmcs_1769 Mmcs_3796 FJO: Fjoh_4612 Mmcs_3864FPS: FP0770 FP1586 FP1725 MKM: Mkms_0251 Mkms_1540 Mkms_1805 RRS:RoseRS_3911 RoseRS_4348 Mkms_1816 Mkms_2836 Mkms_3159 RCA: Rcas_0702Rcas_3206 Mkms_3286 Mkms_3869 Mkms_3938 Mkms_4227 HAU: Haur_0522Mkms_4411 Mkms_4580 DRA: DR_1072 DR_1428 DR_1960 DR_2480 Mkms_4724Mkms_4764 Mkms_4776 DR_A0053 MJL: Mjls_0231 Mjls_1739 Mjls_1750Mjls_2819 DGE: Dgeo_0755 Dgeo_1305 Dgeo_1441 Dgeo_1883 Mjls_3119Mjls_3235 TTH: TTC0191 TTC0330 Mjls_3800 Mjls_3850 Mjls_4110 Mjls_4383TTJ: TTHA0559 Mjls_4705 Mjls_4876 TME: Tmel_1134 Mjls_5018 Mjls_5063Mjls_5075 FNO: Fnod_0314 CGL: NCgl2309(cgl2392) PMO: Pmob_0515 CGB:cg2625(pcaF) HMA: rrnAC0896(acaB3) rrnAC2815(aca2) CEF: CE0731 CE2295rrnAC3497(yqeF) CJK: jk1543(fadA3) rrnB0240(aca1) rrnB0242(acaB2)rrnB0309(acaB1) NFA: nfa10750(fadA4) TAC: Ta0582 RHA: RHA1_ro01455RHA1_ro01623 TVO: TVN0649 RHA1_ro01876 RHA1_ro02517(catF) PTO: PTO1505RHA1_ro03022 RHA1_ro03024 RHA1_ro03391 APE: APE_2108 RHA1_ro03892 SSO:SSO2377(acaB-4) RHA1_ro04599 RHA1_ro05257 RHA1_ro08871 STO: ST0514 SCO:SCO5399(SC8F4.03) SAI: Saci_0963 Saci_1361(acaB1) SMA: SAV1384(fadA5)SAV2856(fadA1) MSE: Msed_0656 ART: Arth_1160 Arth_2986 Arth_3268Arth_4073 PAI: PAE1220 NCA: Noca_1371 Noca_1797 Noca_1828 Noca_2764 PIS:Pisl_0029 Pisl_1301 Noca_4142 PCL: Pcal_0781 TFU: Tfu_1520 Tfu_2394 PAS:Pars_0309 Pars_1071 FRA: Francci3_3687 CMA: Cmaq_1941 FRE: Franean1_1044Franean1_2711 Franean1_2726 Franean1_3929 Franean1_4037 Franean1_4577FAL: FRAAL2514 FRAAL2618 FRAAL5910(atoB) ACE: Acel_0626 Acel_0672 SEN:SACE_1192(mmgA) SACE_2736(fadA6) SACE_4011(catF) SACE_6236(fadA4) STP:Strop_3610 SAQ: Sare_1316 Sare_3991 RXY: Rxyl_1582 Rxyl_1842 Rxyl_2389Rxyl_2530 FNU: FN0495 BGA: BG0110(fadA) BAF: BAPKO_0110(fadA) LIL:LA0457(thiL1) LA0828(thiL2) LA4139(fadA) LIC: LIC10396(phbA) LBJ:LBJ_2862(paaJ-4) LBL: LBL_0209(paaJ-4) SYN: slr1993(phaA) SRU:SRU_1211(atoB) SRU_1547 CHU: CHU_1910(atoB) GFO: GFO_1507(atoB)Exemplary HMG-CoA synthase nucleic acids and polypeptides HSA:3157(HMGCS1) 3158(HMGCS2) MXA: MXAN_3948(tac) MXAN_4267(mvaS) PTR:457169(HMGCS2) 461892(HMGCS1) BSU: BG10926(pksG) MCC: 702553(HMGCS1)713541(HMGCS2) OIH: OB2248 MMU: 15360(Hmgcs2) 208715(Hmgcs1) SAU:SA2334(mvaS) RNO: 24450(Hmgcs2) 29637(Hmgcs1) SAV: SAV2546(mvaS) CFA:479344(HMGCS1) 607923(HMGCS2) SAM: MW2467(mvaS) BTA: 407767(HMGCS1) SAR:SAR2626(mvaS) SSC: 397673(CH242-38B5.1) SAS: SAS2432 GGA: 396379(HMGCS1)SAC: SACOL2561 XLA: 380091(hmgcs1) 447204(MGC80816) SAB: SAB2420(mvaS)DRE: 394060(hmgcs1) SAA: SAUSA300_2484 SPU: 578259(LOC578259) SAO:SAOUHSC_02860 DME: Dmel_CG4311(Hmgs) SAJ: SaurJH9_2569 CEL: F25B4.6 SAH:SaurJH1_2622 ATH: AT4G11820(BAP1) SEP: SE2110 OSA: 4331418 4347614 SER:SERP2122 CME: CMM189C SHA: SH0508(mvaS) SCE: YML126C(ERG13) SSP: SSP0324AGO: AGOS_ADL356C LMO: lmo1415 PIC: PICST_83020 LMF: LMOf2365_1434(mvaS)CAL: CaO19_7312(CaO19.7312) LIN: lin1454 CGR: CAGL0H04081g LWE:lwe1432(mvaS) SPO: SPAC4F8.14c(hcs) LLA: L13187(hmcM) MGR: MGG_01026LLC: LACR_1666 ANI: AN4923.2 LLM: llmg_0929(hmcM) AFM: AFUA_3G10660AFUA_8G07210 SPY: SPy_0881(mvaS.2) AOR: AO090003000611 AO090010000487SPZ: M5005_Spy_0687(mvaS.1) CNE: CNC05080 CNG02670 SPM:spyM18_0942(mvaS2) UMA: UM05362.1 SPG: SpyM3_0600(mvaS.2) ECU:ECU10_0510 SPS: SPs1253 DDI: DDBDRAFT_0217522 DDB_0219924(hgsA) SPH:MGAS10270_Spy0745(mvaS1) TET: TTHERM_00691190 SPI:MGAS10750_Spy0779(mvaS1) TBR: Tb927.8.6110 SPJ: MGAS2096_Spy0759(mvaS1)YPE: YPO1457 SPK: MGAS9429_Spy0743(mvaS1) YPK: y2712(pksG) SPF:SpyM51121(mvaS) YPM: YP_1349(pksG) SPA: M6_Spy0704 YPA: YPA_0750 SPB:M28_Spy0667(mvaS.1) YPN: YPN_2521 SPN: SP_1727 YPP: YPDSF_1517 SPR:spr1571(mvaS) YPS: YPTB1475 SPD: SPD_1537(mvaS) CBD: COXBU7E912_1931SAG: SAG1316 TCX: Tcr_1719 SAN: gbs1386 DNO: DNO_0799 SAK: SAK_1347 BMA:BMAA1212 SMU: SMU.943c BPS: BPSS1002 STC: str0577(mvaS) BPM:BURPS1710b_A2613 STL: stu0577(mvaS) BPL: BURPS1106A_A1384 STE: STER_0621BPD: BURPS668_A1470 SSA: SSA_0338(mvaS) BTE: BTH_II1670 SSU: SSU05_1641SSV: SSU98_1652 OOE: OEOE_0968 SGO: SGO_0244 LME: LEUM_1184 LPL:lp_2067(mvaS) NFA: nfa22120 LJO: LJ1607 SEN: SACE_4570(pksG) LAC:LBA0628(hmcS) BBU: BB0683 LSA: LSA1484(mvaS) BGA: BG0706 LSL: LSL_0526BAF: BAPKO_0727 LDB: Ldb0881(mvaS) FJO: Fjoh_0678 LBU: LBUL_0806 HAL:VNG1615G(mvaB) LBR: LVIS_1363 HMA: rrnAC1740(mvaS) LCA: LSEI_1785 HWA:HQ2868A(mvaB) LGA: LGAS_1372 NPH: NP2608A(mvaB_1) NP4836A(mvaB_2) LRE:Lreu_0676 PPE: PEPE_0868 EFA: EF1363 Exemplary hydroxymethylglutaryl-CoAreductase nucleic acids and polypeptides HSA: 3156(HMGCR) PAT: Patl_0427PTR: 471516(HMGCR) CBU: CBU_0030 CBU_0610 MCC: 705479(HMGCR) CBD:COXBU7E912_0151 MMU: 15357(Hmgcr) COXBU7E912_0622(hmgA) RNO:25675(Hmgcr) TCX: Tcr_1717 CFA: 479182(HMGCR) DNO: DNO_0797 BTA:407159(HMGCR) CVI: CV_1806 GGA: 395145(RCJMB04_14m24) SUS: Acid_5728Acid_6132 SPU: 373355(LOC373355) SAU: SA2333(mvaA) DME:Dmel_CG10367(Hmgcr) SAV: SAV2545(mvaA) CEL: F08F8.2 SAM: MW2466(mvaA)OSA: 4347443 SAB: SAB2419c(mvaA) SCE: YLR450W(HMG2) YML075C(HMG1) SEP:SE2109 AGO: AGOS_AER152W LWE: lwe0819(mvaA) CGR: CAGL0L11506g LLA:L10433(mvaA) SPO: SPCC162.09c(hmg1) LLC: LACR_1664 ANI: AN3817.2 LLM:llmg_0931(mvaA) AFM: AFUA_1G11230 AFUA_2G03700 SPY: SPy_0880(mvaS.1)AOR: AO090103000311 AO090120000217 SPM: spyM18_0941(mvaS1) CNE: CNF04830SPG: SpyM3_0599(mvaS.1) UMA: UM03014.1 SPS: SPs1254 ECU: ECU10_1720 SPH:MGAS10270_Spy0744 DDI: DDB_0191125(hmgA) DDB_0215357(hmgB) SPI:MGAS10750_Spy0778 TBR: Tb927.6.4540 SPJ: MGAS2096_Spy0758 TCR: 506831.40509167.20 SPK: MGAS9429_Spy0742 LMA: LmjF30.3190 SPA: M6_Spy0703 VCH:VCA0723 SPN: SP_1726 VCO: VC0395_0662 SAG: SAG1317 VVU: VV2_0117 SAN:gbs1387 VVY: VVA0625 STC: str0576(mvaA) VPA: VPA0968 STL: stu0576(mvaA)VFI: VFA0841 STE: STER_0620 SSA: SSA_0337(mvaA) HAL: VNG1875G(mvaA) LPL:lp_0447(mvaA) HMA: rrnAC3412(mvaA) LJO: LJ1608 HWA: HQ3215A(hmgR) LSL:LSL_0224 NPH: NP0368A(mvaA_2) NP2422A(mvaA_1) LBR: LVIS_0450 TAC:Ta0406m LGA: LGAS_1373 TVO: TVN1168 EFA: EF1364 PTO: PTO1143 NFA:nfa22110 PAB: PAB2106(mvaA) BGA: BG0708(mvaA) PFU: PF1848 SRU: SRU_2422TKO: TK0914 FPS: FP2341 RCI: RCIX1027(hmgA) RCIX376(hmgA) MMP:MMP0087(hmgA) APE: APE_1869 MMQ: MmarC5_1589 IHO: Igni_0476 MAC:MA3073(hmgA) HBU: Hbut_1531 MBA: Mbar_A1972 SSO: SSO0531 MMA: MM_0335STO: ST1352 MBU: Mbur_1098 SAI: Saci_1359 MHU: Mhun_3004 PAI: PAE2182MEM: Memar_2365 PIS: Pisl_0814 MBN: Mboo_0137 PCL: Pcal_1085 MTH: MTH562PAS: Pars_0796 MST: Msp_0584(hmgA) MSI: Msm_0227 MKA: MK0355(HMG1) AFU:AF1736(mvaA) Exemplary mevalonate kinase nucleic acids and polypeptidesHSA: 4598(MVK) TET: TTHERM_00637680 MCC: 707645(MVK) TBR: Tb927.4.4070MMU: 17855(Mvk) TCR: 436521.9 509237.10 RNO: 81727(Mvk) LMA: LmjF31.0560CFA: 486309(MVK) CBU: CBU_0608 CBU_0609 BTA: 505792(MVK) CBD:COXBU7E912_0620(mvk) GGA: 768555(MVK) LPN: lpg2039 DRE: 492477(zgc:103473) LPF: lpl2017 SPU: 585785(LOC585785) LPP: lpp2022 DME:Dmel_CG33671 BBA: Bd1027(lmbP) Bd1630(mvk) OSA: 4348331 MXA:MXAN_5019(mvk) SCE: YMR208W(ERG12) OIH: OB0225 AGO: AGOS_AER335W SAU:SA0547(mvaK1) PIC: PICST_40742(ERG12) SAV: SAV0590(mvaK1) CGR:CAGL0F03861g SAM: MW0545(mvaK1) SPO: SPAC13G6.11c SAR: SAR0596(mvaK1)MGR: MGG_06946 SAS: SAS0549 ANI: AN3869.2 SAC: SACOL0636(mvk) AFM:AFUA_4G07780 SAB: SAB0540(mvaK1) AOR: AO090023000793 SAA:SAUSA300_0572(mvk) CNE: CNK01740 SAO: SAOUHSC_00577 ECU: ECU09_1780 SEP:SE0361 DDI: DDBDRAFT_0168621 SER: SERP0238(mvk) SHA: SH2402(mvaK1) LCA:LSEI_1491 SSP: SSP2122 LGA: LGAS_1033 LMO: lmo0010 LRE: Lreu_0915 LMF:LMOf2365_0011 PPE: PEPE_0927 LIN: lin0010 EFA: EF0904(mvk) LWE:lwe0011(mvk) OOE: OEOE_1100 LLA: L7866(yeaG) LME: LEUM_1385 LLC:LACR_0454 NFA: nfa22070 LLM: llmg_0425(mvk) BGA: BG0711 SPY:SPy_0876(mvaK1) BAF: BAPKO_0732 SPZ: M5005_Spy_0682(mvaK1) FPS: FP0313SPM: spyM18_0937(mvaK1) MMP: MMP1335 SPG: SpyM3_0595(mvaK1) MAE:Maeo_0775 SPS: SPs1258 MAC: MA0602(mvk) SPH: MGAS10270_Spy0740(mvaK1)MBA: Mbar_A1421 SPI: MGAS10750_Spy0774(mvaK1) MMA: MM_1762 SPJ:MGAS2096_Spy0753(mvaK1) MBU: Mbur_2395 SPK: MGAS9429_Spy0737(mvaK1) MHU:Mhun_2890 SPF: SpyM51126(mvaK1) MEM: Memar_1812 SPA: M6_Spy0699 MBN:Mboo_2213 SPB: M28_Spy0662(mvaK1) MST: Msp_0858(mvk) SPN: SP_0381 MSI:Msm_1439 SPR: spr0338(mvk) MKA: MK0993(ERG12) SPD: SPD_0346(mvk) HAL:VNG1145G(mvk) SAG: SAG1326 HMA: rrnAC0077(mvk) SAN: gbs1396 HWA:HQ2925A(mvk) SAK: SAK_1357(mvk) NPH: NP2850A(mvk) SMU: SMU.181 PTO:PTO1352 STC: str0559(mvaK1) PHO: PH1625 STL: stu0559(mvaK1) PAB:PAB0372(mvk) STE: STER_0598 PFU: PF1637(mvk) SSA: SSA_0333(mvaK1) TKO:TK1474 SSU: SSU05_0289 RCI: LRC399(mvk) SSV: SSU98_0285 APE: APE_2439SGO: SGO_0239(mvk) HBU: Hbut_0877 LPL: lp_1735(mvaK1) SSO: SSO0383 LJO:LJ1205 STO: ST2185 LAC: LBA1167(mvaK) SAI: Saci_2365(mvk) LSA:LSA0908(mvaK1) MSE: Msed_1602 LSL: LSL_0685(eRG) PAI: PAE3108 LDB:Ldb0999(mvk) PIS: Pisl_0467 LBU: LBUL_0906 PCL: Pcal_1835 LBR: LVIS_0858Exemplary phosphomevalonate kinase nucleic acids and polypeptides HSA:10654(PMVK) CFA: 612251(PMVK) PTR: 457350(PMVK) BTA: 513533(PMVK) MCC:717014(PMVK) DME: Dmel_CG10268 MMU: 68603(Pmvk) ATH: AT1G31910 OSA:4332275 SPS: SPs1256 SCE: YMR220W(ERG8) SPH: MGAS10270_Spy0742(mvaK2)AGO: AGOS_AER354W SPI: MGAS10750_Spy0776(mvaK2) PIC: PICST_52257(ERG8)SPJ: MGAS2096_Spy0755(mvaK2) CGR: CAGL0F03993g SPK:MGAS9429_Spy0739(mvaK2) SPO: SPAC343.01c SPF: SpyM51124(mvaK2) MGR:MGG_05812 SPA: M6_Spy0701 ANI: AN2311.2 SPB: M28_Spy0664(mvaK2) AFM:AFUA_5G10680 SPN: SP_0383 AOR: AO090010000471 SPR: spr0340(mvaK2) CNE:CNM00100 SPD: SPD_0348(mvaK2) UMA: UM00760.1 SAG: SAG1324 DDI:DDBDRAFT_0184512 SAN: gbs1394 TBR: Tb09.160.3690 SAK: SAK_1355 TCR:507913.20 508277.140 SMU: SMU.938 LMA: LmjF15.1460 STC: str0561(mvaK2)MXA: MXAN_5017 STL: stu0561(mvaK2) OIH: OB0227 STE: STER_0600 SAU:SA0549(mvaK2) SSA: SSA_0335(mvaK2) SAV: SAV0592(mvaK2) SSU: SSU05_0291SAM: MW0547(mvaK2) SSV: SSU98_0287 SAR: SAR0598(mvaK2) SGO: SGO_0241SAS: SAS0551 LPL: lp_1733(mvaK2) SAC: SACOL0638 LJO: LJ1207 SAB:SAB0542(mvaK2) LAC: LBA1169 SAA: SAUSA300_0574 LSA: LSA0906(mvaK2) SAO:SAOUHSC_00579 LSL: LSL_0683 SAJ: SaurJH9_0615 LDB: Ldb0997(mvaK) SEP:SE0363 LBU: LBUL_0904 SER: SERP0240 LBR: LVIS_0860 SHA: SH2400(mvaK2)LCA: LSEI_1092 SSP: SSP2120 LGA: LGAS_1035 LMO: lmo0012 LRE: Lreu_0913LMF: LMOf2365_0013 PPE: PEPE_0925 LIN: lin0012 EFA: EF0902 LWE: lwe0013NFA: nfa22090 LLA: L10014(yebA) BGA: BG0710 LLC: LACR_0456 BAF:BAPKO_0731 LLM: llmg_0427 NPH: NP2852A SPY: SPy_0878(mvaK2) SSO: SSO2988SPZ: M5005_Spy_0684(mvaK2) STO: ST0978 SPM: spyM18_0939 SAI: Saci_1244SPG: SpyM3_0597(mvaK2) Exemplary diphosphomevalonate decarboxylasenucleic acids and polypeptides HSA: 4597(MVD) CFA: 489663(MVD) PTR:468069(MVD) GGA: 425359(MVD) MCC: 696865(MVD) DME: Dmel_CG8239 MMU:192156(Mvd) SCE: YNR043W(MVD1) RNO: 81726(Mvd) AGO: AGOS_AGL232C PIC:PICST_90752 SPH: MGAS10270_Spy0741(mvaD) CGR: CAGL0C03630g SPI:MGAS10750_Spy0775(mvaD) SPO: SPAC24C9.03 SPJ: MGAS2096_Spy0754(mvaD)MGR: MGG_09750 SPK: MGAS9429_Spy0738(mvaD) ANI: AN4414.2 SPF:SpyM51125(mvaD) AFM: AFUA_4G07130 SPA: M6_Spy0700 AOR: AO090023000862SPB: M28_Spy0663(mvaD) CNE: CNL04950 SPN: SP_0382 UMA: UM05179.1 SPR:spr0339(mvd1) DDI: DDBDRAFT_0218058 SPD: SPD_0347(mvaD) TET:TTHERM_00849200 SAG: SAG1325(mvaD) TBR: Tb10.05.0010 Tb10.61.2745 SAN:gbs1395 TCR: 507993.330 511281.40 SAK: SAK_1356(mvaD) LMA: LmjF18.0020SMU: SMU.937 CBU: CBU_0607(mvaD) STC: str0560(mvaD) CBD:COXBU7E912_0619(mvaD) STL: stu0560(mvaD) LPN: lpg2040 STE: STER_0599LPF: lpl2018 SSA: SSA_0334(mvaD) LPP: lpp2023 SSU: SSU05_0290 TCX:Tcr_1734 SSV: SSU98_0286 DNO: DNO_0504(mvaD) SGO: SGO_0240(mvaD) BBA:Bd1629 LPL: lp_1734(mvaD) MXA: MXAN_5018(mvaD) LJO: LJ1206 OIH: OB0226LAC: LBA1168(mvaD) SAU: SA0548(mvaD) LSA: LSA0907(mvaD) SAV:SAV0591(mvaD) LSL: LSL_0684 SAM: MW0546(mvaD) LDB: Ldb0998(mvaD) SAR:SAR0597(mvaD) LBU: LBUL_0905 SAS: SAS0550 LBR: LVIS_0859 SAC:SACOL0637(mvaD) LCA: LSEI_1492 SAB: SAB0541(mvaD) LGA: LGAS_1034 SAA:SAUSA300_0573(mvaD) LRE: Lreu_0914 SAO: SAOUHSC_00578 PPE: PEPE_0926SAJ: SaurJH9_0614 EFA: EF0903(mvaD) SAH: SaurJH1_0629 LME: LEUM_1386SEP: SE0362 NFA: nfa22080 SER: SERP0239(mvaD) BBU: BB0686 SHA:SH2401(mvaD) BGA: BG0709 SSP: SSP2121 BAF: BAPKO_0730 LMO: lmo0011 GFO:GFO_3632 LMF: LMOf2365_0012(mvaD) FPS: FP0310(mvaD) LIN: lin0011 HAU:Haur_1612 LWE: lwe0012(mvaD) HAL: VNG0593G(dmd) LLA: L9089(yeaH) HMA:rrnAC1489(dmd) LLC: LACR_0455 HWA: HQ1525A(mvaD) LLM: llmg_0426(mvaD)NPH: NP1580A(mvaD) SPY: SPy_0877(mvaD) PTO: PTO0478 PTO1356 SPZ:M5005_Spy_0683(mvaD) SSO: SSO2989 SPM: spyM18_0938(mvd) STO: ST0977 SPG:SpyM3_0596(mvaD) SAI: Saci_1245(mvd) SPS: SPs1257 MSE: Msed_1576Exemplary isopentenyl phosphate kinases (IPK) nucleic acids andpolypeptides Methanobacterium thermoautotrophicum Picrophilus torridusDSM9790 (IG-57) gi|48477569 gi|2621082 Pyrococcus abyssi gi|14520758Methanococcus jannaschii DSM 2661 gi|1590842; Pyrococcus horikoshii OT3gi|3258052 Methanocaldococcus jannaschii gi|1590842 Archaeoglobusfulgidus DSM4304 gi|2648231 Methanothermobacter thermautotrophicusgi|2621082 Exemplary isopentenyl-diphosphate Delta-isomerase (IDI)nucleic acids and polypeptides HSA: 3422(IDI1) 91734(IDI2) STY: STY3195PTR: 450262(IDI2) 450263(IDI1) STT: t2957 MCC: 710052(LOC710052)721730(LOC721730) SPT: SPA2907(idi) MMU: 319554(Idi1) SEC: SC2979(idi)RNO: 89784(Idi1) STM: STM3039(idi) GGA: 420459(IDI1) SFL: SF2875(idi)XLA: 494671(LOC494671) SFX: S3074 XTR: 496783(idi2) SFV: SFV_2937 SPU:586184(LOC586184) SSN: SSON_3042 SSON_3489(yhfK) CEL: K06H7.9(idi-1)SBO: SBO_3103 ATH: AT3G02780(IPP2) SDY: SDY_3193 OSA: 4338791 4343523ECA: ECA2789 CME: CMB062C PLU: plu3987 SCE: YPL117C(IDI1) ENT:Ent638_3307 AGO: AGOS_ADL268C SPE: Spro_2201 PIC: PICST_68990(IDI1) VPA:VPA0278 CGR: CAGL0J06952g VFI: VF0403 SPO: SPBC106.15(idi1) PPR:PBPRA0469(mvaD) ANI: AN0579.2 PEN: PSEEN4850 AFM: AFUA_6G11160 CBU:CBU_0607(mvaD) AOR: AO090023000500 CBD: COXBU7E912_0619(mvaD) CNE:CNA02550 LPN: lpg2051 UMA: UM04838.1 LPF: lpl2029 ECU: ECU02_0230 LPP:lpp2034 DDI: DDB_0191342(ipi) TCX: Tcr_1718 TET: TTHERM_00237280TTHERM_00438860 HHA: Hhal_1623 TBR: Tb09.211.0700 DNO: DNO_0798 TCR:408799.19 510431.10 EBA: ebA5678 p2A143 LMA: LmjF35.5330 DVU:DVU1679(idi) EHI: 46.t00025 DDE: Dde_1991 ECO: b2889(idi) LIP: LI1134ECJ: JW2857(idi) BBA: Bd1626 ECE: Z4227 AFW: Anae109_4082 ECS: ECs3761MXA: MXAN_5021(fni) ECC: c3467 RPR: RP452 ECI: UTI89_C3274 RTY:RT0439(idi) ECP: ECP_2882 RCO: RC0744 ECV: APECO1_3638 RFE: RF_0785(fni)ECW: EcE24377A_3215(idi) RBE: RBE_0731(fni) ECX: EcHS_A3048 RAK:A1C_04190 RBO: A1I_04755 SPZ: M5005_Spy_0685 RCM: A1E_02555 SPM:spyM18_0940 RRI: A1G_04195 SPG: SpyM3_0598 MLO: mlr6371 SPS: SPs1255RET: RHE_PD00245(ypd00046) SPH: MGAS10270_Spy0743 XAU: Xaut_4134 SPI:MGAS10750_Spy0777 SIL: SPO0131 SPJ: MGAS2096_Spy0756 SIT: TM1040_3442SPK: MGAS9429_Spy0740 RSP: RSP_0276 SPF: SpyM51123(fni) RSH:Rsph17029_1919 SPA: M6_Spy0702 RSQ: Rsph17025_1019 SPB: M28_Spy0665 JAN:Jann_0168 SPN: SP_0384 RDE: RD1_0147(idi) SPR: spr0341(fni) DSH:Dshi_3527 SPD: SPD_0349(fni) BSU: BG11440(ypgA) SAG: SAG1323 BAN: BA1520SAN: gbs1393 BAR: GBAA1520 SAK: SAK_1354(fni) BAA: BA_2041 SMU: SMU.939BAT: BAS1409 STC: str0562(idi) BCE: BC1499 STL: stu0562(idi) BCA:BCE_1626 STE: STER_0601 BCZ: BCZK1380(fni) SSA: SSA_0336 BCY:Bcer98_1222 SGO: SGO_0242 BTK: BT9727_1381(fni) LPL: lp_1732(idi1) BTL:BALH_1354 LJO: LJ1208 BLI: BL02217(fni) LAC: LBA1171 BLD: BLi02426 LSA:LSA0905(idi) BAY: RBAM_021020(fni) LSL: LSL_0682 BPU: BPUM_2020(fni)LDB: Ldb0996(fni) OIH: OB0537 LBU: LBUL_0903 SAU: SA2136(fni) LBR:LVIS_0861 SAV: SAV2346(fni) LCA: LSEI_1493 SAM: MW2267(fni) LGA:LGAS_1036 SAR: SAR2431(fni) LRE: Lreu_0912 SAS: SAS2237 EFA: EF0901 SAC:SACOL2341(fni) OOE: OEOE_1103 SAB: SAB2225c(fni) STH: STH1674 SAA:SAUSA300_2292(fni) CBE: Cbei_3081 SAO: SAOUHSC_02623 DRM: Dred_0474 SEP:SE1925 SWO: Swol_1341 SER: SERP1937(fni-2) MTA: Moth_1328 SHA:SH0712(fni) MTU: Rv1745c(idi) SSP: SSP0556 MTC: MT1787(idi) LMO: lmo1383MBO: Mb1774c(idi) LMF: LMOf2365_1402(fni) MBB: BCG_1784c(idi) LIN:lin1420 MPA: MAP3079c LWE: lwe1399(fni) MAV: MAV_3894(fni) LLA:L11083(yebB) MSM: MSMEG_1057(fni) MSMEG_2337(fni) LLC: LACR_0457 MUL:MUL_0380(idi2) LLM: llmg_0428(fni) MVA: Mvan_1582 Mvan_2176 SPY:SPy_0879 MGI: Mflv_1842 Mflv_4187 MMC: Mmcs_1954 TTJ: TTHB110 MKM:Mkms_2000 MJA: MJ0862 MIL: Mjls_1934 MMP: MMP0043 CGL: NCgl2223(cgl2305)MMQ: MmarC5_1637 CGB: cg2531(idi) MMX: MmarC6_0906 CEF: CE2207 MMZ:MmarC7_1040 CDI: DIP1730(idi) MAE: Maeo_1184 NFA: nfa19790 nfa22100 MVN:Mevan_1058 RHA: RHA1_ro00239 MAC: MA0604(idi) SCO: SCO6750(SC5F2A.33c)MBA: Mbar_A1419 SMA: SAV1663(idi) MMA: MM_1764 LXX: Lxx23810(idi) MBU:Mbur_2397 CMI: CMM_2889(idiA) MTP: Mthe_0474 AAU: AAur_0321(idi) MHU:Mhun_2888 PAC: PPA2115 MLA: Mlab_1665 FRA: Francci3_4188 MEM: Memar_1814FRE: Franean1_5570 MBN: Mboo_2211 FAL: FRAAL6504(idi) MTH: MTH48 KRA:Krad_3991 MST: Msp_0856(fni) SEN: SACE_2627(idiB_2) SACE_5210(idi) MSI:Msm_1441 STP: Strop_4438 MKA: MK0776(lldD) SAQ: Sare_4564 Sare_4928 AFU:AF2287 RXY: Rxyl_0400 HAL: VNG1818G(idi) VNG6081G(crt_1) BBU: BB0684VNG6445G(crt_2) VNG7060 VNG7149 BGA: BG0707 HMA: rrnAC3484(idi) SYN:sll1556 HWA: HQ2772A(idiA) HQ2847A(idiB) SYC: syc2161_c NPH:NP0360A(idiB_1) NP4826A(idiA) SYF: Synpcc7942_1933 NP5124A(idiB_2) CYA:CYA_2395(fni) TAC: Ta0102 CYB: CYB_2691(fni) TVO: TVN0179 TEL: tll1403PTO: PTO0496 ANA: all4591 PHO: PH1202 AVA: Ava_2461 Ava_B0346 PAB:PAB1662 TER: Tery_1589 PFU: PF0856 SRU: SRU_1900(idi) TKO: TK1470 CHU:CHU_0674(idi) RCI: LRC397(fni) GFO: GFO_2363(idi) APE: APE_1765.1 FJO:Fjoh_0269 SMR: Smar_0822 FPS: FP1792(idi) IHO: Igni_0804 CTE: CT0257HBU: Hbut_0539 CCH: Cag_1445 SSO: SSO0063 CPH: Cpha266_0385 STO: ST2059PVI: Cvib_1545 SAI: Saci_0091 PLT: Plut_1764 MSE: Msed_2136 RRS:RoseRS_2437 PAI: PAE0801 RCA: Rcas_2215 PIS: Pisl_1093 HAU: Haur_4687PCL: Pcal_0017 DRA: DR_1087 PAS: Pars_0051 DGE: Dgeo_1381 TPE: Tpen_0272TTH: TT_P0067 Exemplary isoprene synthase nucleic acids and polypeptidesGenbank Accession Nos. AY341431 AY316691 AY279379 AJ457070  AY182241

1. A method for producing a fuel constituent from a bioisoprenecomposition comprising chemically transforming a substantial portion ofthe isoprene in the bioisoprene composition to non-isoprene compoundsby: (a) subjecting the bioisoprene composition to heat or catalyticconditions suitable for isoprene dimerization to produce an isoprenedimer and then catalytically hydrogenating the isoprene dimer to form asaturated C10 fuel constituent; or (b) (i) partially hydrogenating thebioisoprene composition to produce an isoamylene, (ii) dimerizing theisoamylene with a mono-olefin selected from the group consisting ofisoamylene, propylene and isobutene to form a dimate and (iii)completely hydrogenating the dimate to produce a fuel constituent. 2.The method of claim 1, wherein at least about 95% of isoprene in thebioisoprene composition is converted to non-isoprene compounds.
 3. Themethod of claim 1, wherein the bioisoprene composition is heated toabout 150° C. to about 250° C. to produce an unsaturated cyclic isoprenedimer and the unsaturated cyclic isoprene dimer is hydrogenatedcatalytically to produce a saturated cyclic isoprene dimer fuelconstituent.
 4. The method of claim 1, wherein the method comprises: (i)contacting the bioisoprene composition with a catalyst for catalyzingcyclo-dimerization of isoprene to produce an unsaturated cyclic isoprenedimer and the unsaturated cyclic isoprene dimer is hydrogenatedcatalytically to produce a saturated cyclic isoprene dimer fuelconstituent.
 5. The method of claim 4, wherein the catalyst forcatalyzing cyclo-dimerization of isoprene comprising a catalyst selectedfrom the group consisting of a nickel catalyst, iron catalysts andchromium catalysts.
 6. The method of claim 1, wherein the step ofpartially hydrogenating the bioisoprene composition comprises contactingthe bioisoprene composition with hydrogen gas and a catalyst forcatalyzing partial hydrogenation of isoprene.
 7. The method of claim 6,wherein the catalyst for catalyzing partial hydrogenation of isoprenecomprises a palladium catalyst.
 8. The method of claim 1, wherein thestep of dimerizing the isoamylene with a mono-olefin comprisescontacting the isoamylene with the mono-olefin in the presence of acatalyst for catalyzing dimerization of mono-olefin.
 9. The method ofclaim 8, wherein the catalyst for catalyzing dimerization of mono-olefincomprises an acid catalyst.
 10. The method of claim 1, furthercomprising purifying the isoprene from the bioisoprene composition priorto chemically transforming the bioisoprene composition to a fuelconstituent.
 11. A system for producing a fuel constituent from abioisoprene composition, wherein a substantial portion of the isoprenein the bioisoprene composition is chemically converted to non-isoprenecompounds, the system comprising a bioisoprene composition and: (a) (i)one or more chemicals capable of dimerizing isoprene in the bioisoprenecomposition or a source of heat capable of dimerizing isoprene in thebioisoprene composition; and (ii) a catalyst capable of hydrogenatingthe isoprene dimer to form a saturated C10 fuel constituent; or (b) (i)a chemical capable of partially hydrogenating isoprene in thebioisoprene composition to produce an isoamylene, (ii) a chemicalcapable of dimerizing the isoamylene with mono-olefins selected from thegroup consisting of isoamylene, propylene and isobutene to form a dimateand (iii) a chemical capable of completely hydrogenating the dimate toproduce a fuel constituent.
 12. The system of claim 11, wherein thebioisoprene composition comprising greater than about 2 mg of isopreneand comprising greater than or about 99.94% isoprene by weight comparedto the total weight of all C5 hydrocarbons in the composition.
 13. Thesystem of claim 11, wherein the one or more chemicals capable ofdimerizing isoprene comprises catalyst for catalyzing cyclo-dimerizationof isoprene comprising a catalyst selected from the group consisting ofruthenium catalysts, nickel catalysts, iron catalysts and chromiumcatalysts.
 14. The system of claim 11, wherein the catalyst forhydrogenating the unsaturated isoprene dimers comprises a catalystselected from the group consisting of palladium catalysts, nickelcatalysts, ruthenium catalysts and rhodium catalysts.
 15. The system ofclaim 11, wherein the chemical capable of partially hydrogenatingisoprene comprises a palladium catalyst.
 16. The system of claim 11,wherein the chemical capable of dimerizing the isoamylene withmono-olefins comprises an acid catalyst.
 17. A fuel compositioncomprising a fuel constituent produced by the method of claim
 1. 18. Thefuel composition of claim 17, wherein the fuel composition issubstantially free of isoprene.
 19. The fuel composition of claim 17,wherein the fuel composition has δ¹³C value which is greater than −22‰or within the range of −32‰ to −24‰.